Antimicrobial compounds and/or modulators of microbial infections and methods of using the same

ABSTRACT

Some embodiments include compounds that can inhibit the growth of bacterial and/or inhibit or reduce microbial infections caused by one or more microorganisms (e.g.,  Pseudomonas aeruginosa  and  Cryptococcus neoformans ) and methods of using these compounds to treat microbial infection and outbreaks and/or to reduce the formation of biofilms. Other embodiments include synthesis of the compounds that can inhibit the growth of one or more microorganisms and/or inhibit or reduce microbial infections.

PRIORITY CLAIM

This application claims the benefit of U.S. Provisional PatentApplication No. 62/487,271, filed Apr. 19, 2017, and U.S. ProvisionalPatent Application No. 62/644,124, filed Mar. 16, 2018, each of which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to compounds that can inhibit thegrowth of a microorganism and/or inhibit or reduce microbial infectionscaused by one or more microorganisms (e.g., Pseudomonas aeruginosa,Cryptococcus neoformans and Candida albicans) and methods of using thesecompounds to treat microbial infection and outbreaks and/or to reducethe formation of biofilms.

BACKGROUND AND SUMMARY

Microbial infections may cause severe suffering and even death. Whilevirtually all humans and animals are susceptible to microbial infectionscertain sub-population are especially vulnerable to such infections.Vulnerable populations of human include the very young, the very old,persons with poorly developed or weakened immune systems or patientsthat already have underlying health challenges such as Cystic Fibrosisthat make them susceptible to certain microbial infections. Since theintroduction of penicillin in the 1940s, antibiotics have served as oneof the major treatments for pathogenic microbial infections.

Unfortunately not all bacteria are susceptible to treatment withantibiotics. Classes of bacteria that are especially pathogenic includebacteria that readily form biofilms. Some of the uses of these biofilmsin the pathogenic setting include helping to anchor bacteria to aportion of the body and to help the bacteria existing in conjunctionwith the biofilm to evade the body's immune system. Moreover, thewidespread use of broad spectrum antibiotics has helped to give rise toantibiotic resistant strains of pathogenic bacteria which were onceeasily controlled with such compounds. Accordingly, there exists a needfor compounds that have the ability to stop or at least slow the growthof pathogenic bacteria.

Some but not necessarily all bacteria that can be effected by thecompounds and or methods disclosed herein form biofilms. The ability ofa compounds and/or methods to reduce or even inhibit the formation ofbiofilms can be used as basis for an assay to determine the efficacy ofsuch compounds as inhibitor so bacteria growth. Although many biofilmsare relatively harmless, some can be very malicious and cause seriousillnesses. They have been associated with urinary tract infections, earinfections, and colonization of implanted medical devices. For example,one culprit which produces biofilms is Pseudomonas aeruginosa (PA). PAis particularly dangerous to those suffering from cystic fibrosis.Cystic fibrosis is a recessively inherited genetic disease caused by amutation in the gene which codes for the protein cystic fibrosistransmembrane conductance regulator (CFTR). The malfunction of CFTRleads to increased mucus accumulation in the lungs and other organs,which is an ideal medium for P. aeruginosa and other bacteria tocolonize into biofilms.

The study of biofilms has afforded a new strategy in combating virulentbacteria, such as P. aeruginosa. Instead of formulating drugs to killharmful bacteria, biofilms can be treated with drugs which signal thebacteria to leave the biofilm and disperse or reduce biofilm formation.Causing the bacteria to disperse comes with the benefit of reducing themto a benign and defenseless state, allowing the body's own immune systemto destroy the invaders without side effects from drugs and withoutinducing antibiotic resistance in the bacteria. Accordingly, the abilityof a drug to inhibit or reduce formation of biofilms of bacteriaprovides an excellent indication that the drug will likely inhibit orreduce the growth of the bacteria.

For more information about biofilm composition and development, seeFlemming, H. C. et al., The Biofilm Matrix. NATURE REVIEWS MICROBIOLOGY2010, 8, 623-633, Hall-Stoodley, L. et al., Bacterial Biofilms: From theNatural Environment to Infectious Diseases. NATURE REVIEWS MICROBIOLOGY2004, 2, 95-108; Kolodkin-Gal, I. et al., D-Amino Acids Trigger BiofilmDisassembly. SCIENCE 2010 328, 627-629.

Aspects of the instant invention include syntheses of compounds that mayinhibit or reduce the growth of microorganism including, but is notlimited to, certain pathogenic bacteria (e.g., Pseudomonas aeruginosa(Pa)) and fungi (e.g. Cryptococcus neoformans (Cn) and Candida albicans(Ca)). Some of these compounds may also reduce or inhibit thedevelopment of biofilms that may contribute to the pathology of certainstrains of microorganism. These compounds include amide derivatives offluorinated phenyl groups, in some instances specific enantiomers ofsuch compounds are especially effective anti-microbial agents. Someembodiments include compounds that exhibit the ability to stop or atleast slow the growth of Pseudomonas aeruginosa (and/or Cryptococcusneoformans), a pathogen known to be responsible for severe microbialinfection in patient who has Cystic Fibrosis.

Some aspects of the invention include methods of synthesizing suchcompounds and using the same to treat microbial infections and toeliminate or at least reduce the formation of biofilms associated withthe growth of certain types of microorganisms (e.g., Pseudomonasaeruginosa, Candida albicans, and Cryptococcus neoformans).

A first embodiment of the present disclosure includes at least onecompound of the following Formula or a pharmaceutically acceptable saltthereof, or a metabolite thereof:

R¹ is selected from the group consisting of: biphenyl,—(CH₂)_(n)-biphenyl, biphenyl ketone, naphthalene, anthracene, benzyloptionally substituted with 1, 2 or 3 halogens, C₁, C₂, C₃, C₄, C₅, or,C₆ alkyl, C₁, C₂, C₃, C₄, C₅, or, C₆ alkoxy, —OH, —NH₂, —NO₂, —CN, or—CF₃, and phenyl optionally substituted with 1, 2 or 3 halogens, C₁, C₂,C₃, C₄, C₅, or, C₆ alkyl, C₁, C₂, C₃, C₄, C₅, or, C₆ alkoxy, —OH, —NH₂,—NO₂, —CN, or —CF₃;

R² is selected from the group consisting of: —OH, —NHR⁴, and —NR⁴R⁵;

R³ is selected from the group consisting of: —NH₂ and NH—C(O)CR⁶R⁷

R⁴ and R⁵ are independently selected from the group consisting of: H,halogen, C₁, C₂, C₃, C₄, C₅, or, C₆ alkyl being unbranched, branched orcyclic, C₁, C₂, C₃, C₄, C₅, or, C₆ alkoxy, C₁, C₂, C₃, C₄, C₅, or, C₆haloalkoxy, hydroxyl, acyl, acyl amides, carboxyl, tetrazolyl, and—(CH₂)_(n)—R⁸;

Alternatively, R⁴ and R⁵ are taken together to form a pyridine, apiperidine, a pyrrole, or a pyrrolidine ring optionally substituted withC₁, C₂, C₃, C₄, C₅, or, C₆ alkyl, C₁, C₂, C₃, C₄, C₅, or, C₆ alkoxy, orcarboxyl;

R⁶ and R⁷ are independently selected from the group consisting of: H,—NH₂, NHR¹², benzyl optionally substituted with 1, 2 or 3 halogens, C₁,C₂, C₃, C₄, C₅, or, C₆ alkyl, C₁, C₂, C₃, C₄, C₅, or, C₆ alkoxy, —OH,—NH₂, —NO₂, —CN, or —CF₃, and phenyl optionally substituted with 1, 2 or3 halogens, C₁, C₂, C₃, C₄, C₅, or, C₆ alkyl, C₁, C₂, C₃, C₄, C₅, or, C₆alkoxy, —OH, —NH₂, —NO₂, —CN, or —CF₃, C₁, C₂, C₃, C₄, C₅, or, C₆ alkylbeing unbranched, branched or cyclic, C₁, C₂, C₃, C₄, C₅, or, C₆ alkoxy,C₁, C₂, C₃, C₄, C₅, or, C₆ haloalkoxy, hydroxyl, acyl, acyl amides,carboxyl, tetrazolyl, and —(CH₂)_(n)—R⁹;

Alternatively, R⁶ and R⁷ are taken together to form a pyridine, apiperidine, a pyrrole, or a pyrrolidine ring optionally substituted withC₁, C₂, C₃, C₄, C₅, or, C₆ alkyl, C₁, C₂, C₃, C₄, C₅, or, C₆ alkoxy, orcarboxyl;

R⁸ is —OH, —CF₃, morpholinyl, pyridinyl, benzyl optionally substitutedwith 1, 2 or 3 halogens, C₁, C₂, C₃, C₄, C₅, or, C₆ alkyl, C₁, C₂, C₃,C₄, C₅, or, C₆ alkoxy, —OH, —NH₂, —NO₂, —CN, or —CF₃, or phenyloptionally substituted with 1, 2 or 3 halogens, C₁, C₂, C₃, C₄, C₅, or,C₆ alkyl, C₁, C₂, C₃, C₄, C₅, or, C₆ alkoxy, —OH, —NH₂, —NO₂, —CN, or—CF₃;

R⁹ is indole, pyrrole, morpholinyl, pyridinyl, imidazole, guanidyl,C(O)NH₂, benzyl optionally substituted with 1, 2 or 3 halogens, C₁, C₂,C₃, C₄, C₅, or, C₆ alkyl, C₁, C₂, C₃, C₄, C₅, or, C₆ alkoxy, —OH, —NH₂,—NO₂, —CN, or —CF₃, or phenyl optionally substituted with 1, 2 or 3halogens, C₁, C₂, C₃, C₄, C₅, or, C₆ alkyl, C₁, C₂, C₃, C₄, C₅, or, C₆alkoxy, —OH, —NH₂, —NO₂, —CN, or —CF₃, SR¹⁴, or —NHCR¹⁰R¹¹;

R¹⁰ and R¹¹ are independently selected from the group consisting of: H,NH, and NH₂;

R¹² is C₁-C₆ alkyl or C(O)R¹³;

R¹³ is C₁-C₆ alkyl or aryl; and

R¹⁴ is at least one of hydrogen, C₁-C₆ alkyl, or aryl; and

n is 1, 2, 3, or 4.

A second embodiment includes the compound of the first embodiment,wherein: R¹ is benzyl substituted with 1, 2 or 3 halogens, —NH₂, —NO₂,—CN, or —CF₃; R² is —OH; and R³ is NH—C(O)CR⁶R⁷, or a pharmaceuticallyacceptable salt thereof, or a metabolite thereof.

A third embodiment includes the compound of the first and the secondembodiments, wherein the compound is at least one enantiomer of at leastone compound selected from the group consisting of:

or a pharmaceutically acceptable salt thereof, or a metabolite thereof.Consistent with these embodiments, certain embodiments include at leastone compound selected from the compounds represented in FIGS. 14-22,24-28, 30-33, and 35-53.

A fourth embodiment includes the compound of any of the first and thesecond embodiments, wherein the compound is at least one enantiomer ofat least one compound selected from the group consisting of:

or a pharmaceutically acceptable salt thereof, or a metabolite thereof.

A fifth embodiment includes the compound of any of the first and thesecond embodiments, wherein the compound is selected from the groupconsisting of:

or a pharmaceutically acceptable salt thereof, or a metabolite thereof.

A sixth embodiment includes the compound of any of the first and thesecond embodiments, wherein the compound is selected from the groupconsisting of:

a pharmaceutically acceptable salt thereof, or a metabolite thereof.

A seventh embodiment includes a method for reducing the growth of one ormore microorganisms (e.g., bacteria, fungus, and/or yeast), comprisingthe steps of: treating one or more microorganisms with at least onecompound selected from the compounds of the first to the sixth andtwentieth to twenty first embodiments.

An eighth embodiment includes the seventh embodiment, wherein thebacteria are gram-negative bacteria.

A ninth embodiment includes at least one method according to the seventhand the eighth embodiments, wherein the one or more microorganisms caninclude Pseudomonas aeruginosa, Candida albicans, and/or Cryptococcusneoformans.

A tenth embodiment includes at least one method according to any of theseventh to the ninth embodiments, further comprising the step oftreating an area that has been infected by the one or moremicroorganisms. Consistent with these embodiments, the one or moremicroorganisms includes, but is not limited to, bacteria, fungi, and/oryeast.

An eleventh embodiment includes at least one method according to any ofthe seventh to the tenth embodiments, wherein the area comprisessurfaces or hair of an animal, a human, or a plant.

A twelfth embodiment includes a method of treating microbial infections,comprising the steps of: providing to a patient at least onetherapeutically effective dose of at least one compound selected fromthe compounds of the first to the sixth and twentieth to twenty firstembodiments.

A thirteenth embodiment includes the twelfth embodiment, furthercomprising the step of: diagnosing a patient with microbial infections,wherein the microbial infections can be caused by bacteria and/or fungi.Consistent with these embodiments, bacteria and fungi can include, butare not limited to, Pseudomonas aeruginosa, Candida albicans, andCryptococcus neoformans.

A fourteenth embodiment includes the method according to the twelfthembodiment and the thirteenth embodiment, wherein the therapeuticallyeffective dose of the compound selected from the compounds of the firstto the sixth and twentieth to twenty first embodiments is on the orderof between about 1 mg/kg to about 7 mg/kg and the dose of the compoundis administered to the patient at least once per day.

A fifteenth embodiment includes the method according to the twelfthembodiment to the fourteenth embodiments, wherein the therapeuticallyeffective dose of the compound selected from the compounds of the firstto the sixth and twentieth to twenty first embodiments is on the orderof between about 3 mg/kg to about 5 mg/kg and the dose of the compoundis administered to the patient at least once per day.

A sixteenth embodiment includes the method according to the twelfthembodiment to the fifteenth embodiments, wherein the therapeuticallyeffective dose of the compound selected from the compounds of the firstto the sixth and twentieth to twenty first embodiments is administeredby intravenous or intramuscular injections.

A seventeenth embodiment includes at least one compound selected fromthe compounds represented in FIGS. 14-22, 24-28, 30-33, and 35-53.

An eighteenth embodiment includes a method for reducing the growth ofone or more microorganisms, comprising the steps of: treating one ormore microorganisms with at least one compound selected from thecompounds of the seventeenth embodiment. In accordance to thisembodiment, one or more microorganisms include, but is not limited to,Pseudomonas aeruginosa, Candida albicans, and Cryptococcus neoformans.

A nineteenth embodiment includes a method of treating microbialinfections, comprising the steps of: providing to a patient at least onetherapeutically effective dose of at least one compound selected fromthe compounds of the seventeenth embodiment. In accordance to thisembodiment, the microbial infections can be caused by Pseudomonasaeruginosa, Candida albicans, and/or Cryptococcus neoformans.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Experimental design and equipment.

FIG. 2. Various compounds and their corresponding chemical structures.

FIG. 3. Various compounds and their corresponding chemical structures.

FIG. 4. Various compounds and their corresponding chemical structures.

FIG. 5. Various compounds and their corresponding chemical structures.

FIG. 6. Nuclear Magnetic Resonance Spectroscopy (NMR) analysis showingcompounds comprised of a mixture of the two possible diastereomers.

FIG. 7. Graph illustrating an average purity using liquidchromatography/mass spectrometry (LC/MS).

FIG. 8. Photographs of Thin Layer Chromatograph (TLC) showing theseparations.

FIG. 9. Graph showing average percent purity of crude A1 controls.

FIG. 10. Graph showing average masses of crude A1 controls.

FIG. 11. Graph showing average masses of pure A1.

FIG. 12. Photographs of Thin Layer Chromatograph (TLC) plate showingpartial separation of the A1 diastereomers.

FIG. 13. Table showing percent purities of crude samples A2-B3 for all20 experiments. The majority (38 out of 45 duplicates, minus T9 & T10)show excellent reproducibility.

FIG. 14. Table showing various compounds with their correspondingchemical structures, percent inhibition, and IC₅₀.

FIG. 15. Table showing various compounds with their correspondingchemical structures, percent inhibition, and IC₅₀.

FIG. 16. Table showing various compounds with their correspondingchemical structures, percent inhibition, and IC₅₀.

FIG. 17. Table showing various compounds with their correspondingchemical structures, percent inhibition, and IC₅₀.

FIG. 18. Table showing various compounds with their correspondingchemical structures, percent inhibition, and IC₅₀.

FIG. 19. Table showing various compounds with their correspondingchemical structures, percent inhibition, and IC₅₀.

FIG. 20. Table showing various compounds with their correspondingchemical structures, percent inhibition, and IC₅₀.

FIG. 21. Table showing various compounds with their correspondingchemical structures, percent inhibition, and IC₅₀.

FIG. 22. Table showing various compounds with their correspondingchemical structures, percent inhibition, and IC₅₀.

FIG. 23. Representative graphs showing a sample structure with IC₅₀ andR² values.

FIG. 24. Various compounds and their corresponding chemical structures.

FIG. 25. Various compounds and their corresponding chemical structures.

FIG. 26. Various compounds and their corresponding chemical structures.

FIG. 27. Various compounds and their corresponding chemical structures.

FIG. 28. Various compounds and their corresponding chemical structures.

FIG. 29. Assay for Pa biofilm formation inhibition.

FIG. 30. Structure/Activity Data for Active Unnatural Dipeptides.

FIG. 31. Diastereomers Selected for Separation and Biological Screening.

FIG. 32. Preliminary and Hit Confirmation Results for SeparatedDiastereomers.

FIG. 33. General Structure of Targeted Unnatural Dipeptides I and II.

FIG. 34. Bill-Board Position of Reaction Vessels.

FIG. 35. Six Combinatorial Products Made in Hypothetical SingleBill-Board.

FIG. 36. Exemplary applications of the synthetic methods.

FIG. 37. Possible chemical variation and stereochemical combinations.

FIG. 38. Synthesis of isomers as diastereomeric mixture.

FIG. 39. Synthesis of isomers as single compounds.

FIG. 40. Synthesis of isomers as diastereomeric mixture.

FIG. 41. Synthesis of isomers as single compounds.

FIG. 42. Synthesis of isomers as diasteremeric mixture.

FIG. 43. Synthesis of isomers as simpler mixture.

FIG. 44. Possible chemical variation and stereochemical combinations.

FIG. 45. Synthesis of isomers as diastereomeric mixture.

FIG. 46. Synthesis of isomers as single compounds.

FIG. 47. Synthesis of isomers as diastereomeric mixture.

FIG. 48. Synthesis of isomers as single compounds.

FIG. 49. Synthesis of isomers as diastereomeric mixture.

FIG. 50. Synthesis of isomers as two isomeric products.

FIG. 51. Table illustrating percentile (50%) values of MIC, CC₅₀(cytotoxicity) and HC₁₀ (haemolytic activity) for each organism. Unit:μg/mL.

FIG. 52. Data showing various compounds and their activities, tested at32 μg/mL.

FIG. 53. Data showing various compounds and their activities, tested at32 μg/mL.

DESCRIPTION

For the purposes of promoting an understanding of the principles of thenovel technology, reference will now be made to the preferredembodiments thereof, and specific language will be used to describe thesame. It will nevertheless be understood that no limitation of the scopeof the novel technology is thereby intended, such alterations,modifications, and further applications of the principles of the noveltechnology being contemplated as would normally occur to one skilled inthe art to which the novel technology relates are within the scope ofthis disclosure and the claims.

As used herein, unless explicitly stated otherwise or clearly impliedotherwise the term ‘about’ refers to a range of values plus or minus 10percent, e.g. about 1.0 encompasses values from 0.9 to 1.1.

As used herein, unless explicitly stated otherwise or clearly impliedotherwise the meaning of the terms “treatment” or “treating” used inconjunction with various compounds and methods disclosed and claimedherein include, but are not limited to, applying certain compounds tovarious either wet or dry surfaces including, but not limited to theskin, hair or fur of animals or the outside of seeds or plants includingleaves, stems, shoots, roots, branches, blooms, fruits and the like, orany inanimate object either directly or indirectly. The terms“treatment” or “treating” also includes adding compounds to liquids,either aqueous or non-aqueous or mixtures thereof including simplemixtures of such or emulsions. The terms “treatment” or “treating” usedin also include administering compounds to plants, cells, animals andhumans. The terms “treatment” or “treating” include but are not limitedto contacting one or more microorganisms or the biofilms of bacteria,directly or indirectly and may affect the growth of one or moremicroorganisms, and either directly or indirectly the formation ofbacteria biofilms. The terms “treatment” or “treating” as used hereinmay include administering a ‘therapeutically effective’ dose or doses ofcompounds.

As used herein, unless explicitly stated otherwise or clearly impliedotherwise the terms ‘therapeutically effective dose,’ ‘therapeuticallyeffective amounts,’ and the like, refers to a portion of a compound thathas a net positive effect on the health and wellbeing of a human orother animal. Therapeutic effects may include an improvement inlongevity, quality of life and the like these effects also may alsoinclude a reduced susceptibility to developing disease or deterioratinghealth or well-being. The effects may be immediately realized after asingle dose and/or treatment or they may be cumulatively realized aftera series of doses and/or treatments.

Pharmaceutically acceptable salts include salts of compounds of theinvention that are safe and effective for use in mammals and thatpossess a desired therapeutic activity. Pharmaceutically acceptablesalts include salts of acidic or basic groups present in compounds ofthe invention. Pharmaceutically acceptable acid addition salts include,but are not limited to, hydrochloride, hydrobromide, hydroiodide,nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate,acetate, lactate, salicylate, citrate, tartrate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate,trifluoroacetic acid, and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Certain compounds ofthe invention may form pharmaceutically acceptable salts with variousamino acids. Suitable base salts include, but are not limited to,aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, anddiethanolamine salts. For addition information on some pharmaceuticallyacceptable salts that can be used to practice the invention see reviewssuch as Berge, et al., 66 J. PHARM. SCI. 1-19 (1977), and Haynes, et al,J. Pharma. Sci., Vol. 94, No. 10, October 2005, pgs. 2111-2120.

Bacteria are known to communicate via small molecules. Through thiscommunication they are able to create complex, highly-organizedcommunities responsible for biofilm formation, antibiotic resistance,and other important processes. These biofilms are involved in manydisease states, including cystic fibrosis, which is a geneticallyinherited disease affecting approximately 70,000 people worldwide. Dr.Richard Losick and colleagues at Harvard have reported that certainD-amino acids (1, R¹=naturally occurring amino acid side chains in the Dconfiguration) are a trigger for the disassembly of bacterial biofilms(Science, 2010, 328, 627-629). D-Tyrosine was reported to beparticularly active:

EXPERIMENTS AND RESULTS

Referring now to FIG. 1, each section consisted of 5 unique Bill-Boardgrids duplicated for a total of ten. Alkylating agents were distributedacross rows, acylating agents down columns. Each section prepareddifferent controls in A1 and B1.

Masses of crude products ranged from 14-24 mg in most cases (averagepercent yield, 87%) with an average LC/MS purity of 88%. Referring nowto FIG. 8, TLC analysis corroborated the purity results. Goodreproducibility was demonstrated by: (1) the average mass of crude A1controls (T1-T10, 16.6±1.0 mg and T11-T20, 16.2±1.9 mg), (2) the averagepercent purity of crude A1 controls (T1-T10, 95±2.1% and T11 l-T20,92±5.4%) (FIGS. 9-11), and (3) the similar percent purities of 38 out of45 replicate pairs of A2-B3 samples. Average masses of the purified A1controls were 4.8 mg and 4.9 mg (˜38% yield over 5 steps, 82%yield/step) for the two sections and NMR analysis in all cases showedexcellent quality material comprised of a mixture of the two possiblediastereomers (FIG. 6). Cleavage of resin 4 afforded unnatural aminoacids which demonstrated activity against Pseudomonas aeruginosa.

Dipeptides presented in FIGS. 1-4 may be considered “pro-drugs” byvirtue of their potential susceptibility to peptidases. Activity ofdipeptides may be “tunable” by modification of the N-terminus aminoacid. These dipeptide products are a mixture of two possiblediastereomers whose biological activities may likely be different.

The unique molecules prepared by this process are evaluated in biofilmand antimicrobial assays at IUPUI and the University of Queensland,Australia (CO-ADD).

Briefly, CO-ADD, The Community for Open Antimicrobial Drug Discovery, isa global open-access screening initiative launched in February 2015 touncover significant and rich chemical diversity held outside ofcorporate screening collections. For screening, about 1 mg of purecompound is required and the compounds must be soluble in DMSO. In thepreliminary screening, compounds are tested against some ESKAPEpathogens, including, but are not limited to, E. coli, K. pneumoniae, A.baumannii, P. aeruginosa, S. aureus (MRSA), as well as the fungi, forexample, C. neoformans and C. albicans. The screen uses a singleconcentration (e.g., 32 g/mL) and provides initial activity data toselect compounds for further more detailed screening. Active compoundsfrom the preliminary screening are tested in dose response antimicrobialassays to confirm their activity. Active compounds are screened foradverse effects, such as cytotoxicity, critical micelle concentrationand membrane depolarization, as well as for their purity. Then, the hitcompound is tested against a broader panel of microbes withmultidrug-resistant (MDR) and pan-resistant bacterial strains andclinical isolates, with different co-factors (such as serum or lungsurfactant). The hit validation includes initial ADMET screening,including haemolysis, microsome and plasma stability and proteinbinding. See CO-ADD website for more details.

Fmoc-Gly-Wang resin (8.71 g, 6.10 mmol) was swelled for 30 minutes with70 mL of NMP (N-methyl pyrrolidinone) in a 250-mL solid-phase peptidesynthesis vessel under dry argon gas. The vessel was drained and theresin was treated with 35 mL of 20% piperidine in NMP for 2 minutes. Thevessel was drained and the resin was treated with 85 mL of 20%piperidine and was rocked for 45 minutes on an orbital shaker. Thevessel was drained, the resin was washed with 5×70 mL×2 min NMP. Thedeprotected resin was then treated with 10.24 mL of benzophenone iminein 50 mL of NMP followed by 3.04 mL of acetic acid in 50 mL of NMP. Thevessel was rocked overnight at room temperature. After 21 h the vesselwas drained and the resin was washed with 3×65 mL×2 min NMP and 4×65mL×2 min dichloromethane each. The resin 1 was dried under a slow streamof dry nitrogen gas for approximately 30 h and was then stored at 2° C.

50 μmols of resin 1 was treated with 100 μmols of 0.20 M BTPP(t-butylimino-tri(pyrrolidino)phosphorane in NMP followed by 100 μmolsof 0.20 M fluorinated benzyl bromide (R¹X) in NMP. After 2-5 days thereaction mixture was filtered and the resulting resin 2 was washed oncewith 3 mL of tetrahydrofuran (THF). To the resin was then added 2.5 mLof 1.0 N hydrochloric acid (HCl) in THF (1:2). After 20 minutes theresin was filtered and was washed with 3 mL of THF followed by 2×2.5mL×5 min of 0.20 M diisopropylethylamine in NMP, and 2×2.5 mL of NMP togive resin 4. Resins 4 were treated with 250 μmols each of aBoc-protected amino acid and hydroxybenzotriazole (HOBt) (0.25M each inNMP) followed by 250 μmol of 0.50M diisopropylcarbodiimide (DIC) in NMP.After standing 2-5 days the resins were filtered and washed with 3×2 mLeach of NMP and THF and 3×2 mL of dichloromethane to give resins 5.Treatment of resins 5 with 2 mL of 35:60:5 TFA/DCM/H₂O (trifluoroaceticacid/dichloromethane/H₂O) for 30 minutes (drip cleavage) was followed bywashing the resin with 2 mL of 35:60:5 TFA/DCM/H₂O and 2 mL of DCM(dichloromethane). The combined filtrates were evaporated to give crudesalts 6H⁺ which were chromatographed on silica gel usingisopropanol/methanol/ammonium hydroxide mobile phases to elute the freebases of 6H⁺.

Resins 4 were prepared as described in Scheme 1. Resins 4 were treatedwith 250 μmols each of an Fmoc-protected amino acid and HOBt (0.25M eachin NMP) followed by 250 μmol of 0.50M diisopropylcarbodiimide in NMP.After standing 2-5 days the resins were filtered and washed with 4×2 mLof NMP to give resins 7 which were deprotected with 4×2 mL×5 min 20%piperidine in NMP. The resins were then washed with 4×2 ml of NMP, 3×2mL of THF, and 4×2 ml of DCM. To the deprotected resins 8 was then added2 mL of 35/65/5 TFA/DCM/water. The vessels were rotated for 35 minutesat room temperature and were drained into tared vials. The resins werewashed with 2 mL of 35/65/5 TFA/DCM/water and once with 2 mL of DCM. Thefiltrates were then evaporated dryness to give crude salts 9H⁺ whichwere chromatographed on silica gel using isopropanol/methanol/ammoniumhyroxide mobile phases to elute the free bases of 9H⁺.

Fmoc-Ala-Wang resin (74.6 mg, 49.7 μmol) was swelled in 2 mL of NMP forten minutes and then drained. The resin was washed 2 times with 2 mL ofNMP. The resin was then treated 4 times with 2 mL of 20% piperidine inNMP for five minutes each, followed by 4×2 mL×2 min washes with NMP.Resin 10 was then treated with a 0.5M solution of DIC (5 equiv.) in NMPand a 0.25M solution of HOBt and Boc-DL-Phe(4-F)—OH (5 equiv. each) andwas allowed to stand for two days. Resin 11 was washed with 3×2 mL ofNMP, 3×2 mL of THF, and 5×2 mL with DCM. The resin was then treated with2.5 mL of 35:60:5 TFA/DCM/H2O (drip cleavage) for 30 minutes followed byone wash with 2 mL of 35:60:5 TFA/DCM/H2O and 2 mL of DCM. The filtrateswere collected and evaporated to dryness to give the crude salt 26H⁺,which was chromatographed on silica gel usingisopropanol/methanol/ammonium hyroxide mobile phases to elute the freebase of 26H⁺.

LC/MS Analyses

Method A:

Performed using an Agilent Technologies 1200 Series HPLC (HighPerformance Liquid Chromatography) fitted with an Eclipse XDB-C185-micron column, 4.6×150 mm length, 5-microliter injections at a flowrate of 1.0 mL/min. A linear gradient from 20% 1:1 MeCN:MeOH (5 mMNH₄OAc) and 80% water (5 mM NH4OAc) to 100% 1:1 MeCN:MeOH (5 mM NH₄OAc)over 10 minutes was used. Diode array detection (DAD) was performed at210, 214, and 254 nm. Mass spectral analysis was performed on an AgilentTechnologies 6130 Quadrupole LCMS using the electrospray-atmosphericpressure ionization method (ES-API) in the positive mode.

Method B:

Performed using a Kinetex 2.6μ XB-C18 50×2.1 mm column at 50° C., 1.0mL/min, A: 0.1% formic acid in water; B: 0.1 formic acid inacetonitrile; 0.2 min at 5% B, 5-100% B in 3.0 min. Hold 0.5 min at 100%B.

Method C:

Performed using a 3.5μ Waters X-Bridge C18 2.1×50 mm column at 50° C.,1.0 mL/min, A: 10 mM ammonium bicarbonate pH10; B: acetonitrile; 0.2 minhold at 5% B 5-100% B in 3.0 min, hold 0.5 min at 100% B.

(2R,2S)-[2-((S)-2-aminopropanamido)]-3-(4-fluorophenyl)propanoic acid(25)

Method B, 19.0 mg of the trifluoroacetic acid salt of 25, LC/MS, MethodA, (R_(t), m+1/z, purity), 2.90 min, 255, 38% and 3.70 min, 255, 36%;¹HNMR (CD₃OD) δ 1.27 (d, 3H, J=6.9 Hz) and 1.51 (d, 3H, J=7.0 Hz), 2.95(dd, 1H, J=14.1 and 9.9 Hz) and 3.01 (dd, 1H, J=14.2 and 9.3 Hz), 3.26(dd, 1H, J=14.2 and 4.8 Hz), and 3.31 (m, 1H), 3.90 (p, 2H, J=6.4 Hz),4.68 (dd, 1H, J=9.2 and 4.9 Hz) and 4.76 (dd, 1H, J=9.8 and 4.7 Hz),7.03 (t, 4H, J=8.6 Hz), 7.25-7.29 (m, 4H). The crude sample was purifiedby chromatography on silica gel (500 mg) using 18:2:1isopropanol:methanol:concentrated ammonium hydroxide as the mobile phaseto give 7.5 mg (64%), ¹HNMR (CD₃OD) δ 1.25 (d, 3H, J=7.0 Hz) and 1.49(d, 3H, J=6.1 Hz), 2.89 (dd, 1H, J=14.0 and 9.9 Hz) and 2.99 (dd, 1H,J=13.9 and 8.2 Hz), 3.23 (dd, 1H, J=14.0 and 4.7 Hz) and 3.31 (m, 1H),3.87 (br q, 2H, J=6.9 Hz), 4.47 (dd, 1H, J=8.1 and 4.8 Hz) and 4.54 (dd,1H, J=9.5 and 4.4 Hz), 6.96-7.00 (m, 4H), 7.24-7.28 (m, 4H).

(2-amino-[(2R,2S)-3-(4-fluorophenyl)propanoyl)]-L-alanine (26)

Method C, 5.7 mg (45%) of the trifluoroacetic acid salt of 26, LC/MS,Method A, (R_(t), m+1/z, purity), 3.45 min, 255, 45%, 4.74 min, 255,55%.

(2R,2S)-[2-((S)-2-amino-3-methylbutanamido)]-3-(4-fluorophenyl)propanoicacid (27)

Method B, 21.2 mg (106%) of the trifluoroacetic acid salt of 27, LC/MS,Method A, (R_(t), m+1/z, purity), 7.27 min, 283, 37%, 8.39 min, 283,34%.

(2R,2S)-[2-((S)-2-amino-3-(4-hydroxyphenyl)propanamido)]-3-(4-fluorophenyl)propanoicacid (28)

Method B, 24.1 mg (105%) of the trifluoroacetic acid salt of 28, LC/MS,Method A, (R_(t), m+1/z, purity), 3.79 min, 347, 37%, 4.94 min, 347,38%.

Scheme 3: Alternate Preparation of 4a-4d.

(2R,2S)-2-[((2S,3S)-2-amino-4-methylpentanamido)]-3-(4-fluorophenyl)propanoicacid (29)

Method B, 20.0 mg (98%) of the trifluoroacetic acid salt of 29, LC/MS,Method A, (R_(t), m+1/z, purity), 5.54 min, 297, 43%, 6.81 min, 297,46%.

(2R,2S)-[2-(2-aminoacetamido)]-3-(4-fluorophenyl)propanoic acid (30)

Method B, 23.3 mg (132%) of the trifluoroacetic acid salt of 30, LC/MS,Method A, (R_(t), m+1/z, purity), 2.76 min, 241, 92%.

(2R,2S)-[2-((S)-2-amino-3-(4-hydroxyphenyl)propanamido)]-3-(2-fluorophenyl)propanoicacid (31)

Method A, 22.6 mg (98%) of the trifluoroacetic acid salt of 31, LC/MS,Method B, (R_(t), m+1/z, purity), 0.65 min, 347, 52%, 0.74 min, 347,47%.

(2R,2S)-[2-((2S,3S)-2-amino-3-methylpentanamido)]-3-(2-fluorophenyl)propanoicacid (32)

Method A, 23.8 mg (116%) of the trifluoroacetic acid salt of 32, LC/MS,Method A, (R_(t), m 1 z, purity), 5.44 min, 297, 41%, 6.65 min, 297,49%.

(2R,2S)-[2-(2-aminoacetamido)]-3-(2-fluorophenyl)propanoic acid (33)

Method A, 14.2 mg (80%) of the trifluoroacetic acid salt of 33, LC/MS,Method C, (R_(t), m+1/z, purity), 0.31 min, 241, 91%.

(2R,2S)-[2-((R)-2-aminopropanamido)]-3-(4-fluorophenyl)propanoic acid(34)

Method A, 16.6 mg (90%) of the trifluoroacetic acid salt of 34, LC/MS,Method C, (R_(t), m 1/z, purity), 0.54 min, 255, 100%.

(2R,2S)-[2-((S)-2-amino-3-phenylpropanamido)]-3-(4-fluorophenyl)propanoicacid (35)

Method A, 19.0 mg (86%) of the trifluoroacetic acid salt of 35, LC/MS,Method B, (R_(t), m+1/z, purity), 0.80 min, 331, 54% and 0.97 min, 331,46%.

(2R,2S)-[2-((S)-2-aminopropanamido)-3-(2-fluorophenyl)]propanoic acid(36)

Method A, 17.5 mg (95%) of the trifluoroacetic acid salt of 36, LC/MS,Method C, (R_(t), m+1/z, purity), 0.50 min, 255, 100%.

(2R,2S)-[2-((R)-2-aminopropanamido)]-3-(2-fluorophenyl)propanoic acid(37)

Method A, 17.5 mg (95%) of the trifluoroacetic acid salt of 37, LC/MS,Method C, (R_(t), m+1/z, purity), 0.49 min, 255, 95%.

(2R,2S)-[2-((S)-2-amino-3-phenylpropanamido)]-3-(2-fluorophenyl)propanoicacid (38)

Method A, 17.9 mg (81%) of the trifluoroacetic acid salt of 38, LC/MS,Method B, (R_(t), m+1/z, purity), 0.78 min, 331, 52%, 0.95 min, 331,48%.

(2R,2S)-[2-((S)-2-amino-4-methylpentanamido)]-3-(4-fluorophenyl)propanoicacid (39)

Method A, 18.3 mg (89%) of the trifluoroacetic acid salt of 39, LC/MS,Method B, (R_(t), m+1/z, purity), 0.76 min, 297, 43% and 0.93 min, 297,40%.

(2R,2S)-[2-((S)-2-amino-4-methylpentanamido)]-3-(2-fluorophenyl)propanoicacid (40)

Method A, 19.6 mg (96%) of the trifluoroacetic acid salt of 40, LC/MS,Method B, (R_(t), m+1/z, purity), 0.76 min, 297, 51%, 0.90 min, 297,49%.

(2R,2S)-[2-((S)-2-amino-3-(1H-indol-3-yl)propanamido)]-3-(4-fluorophenyl)propanoicacid (41)

Method A, 9.4 mg (51%) of the trifluoroacetic acid salt of 41, LC/MS,Method C, (R_(t), m+1/z, purity), 0.99 min, 370, 85%.

(2R,2S)-[2-((S)-2-amino-3-(1H-indol-3-yl)propanamido)]-3-(3-fluorophenyl)propanoicacid (42)

Method A, 9.3 mg (50%) of the trifluoroacetic acid salt of 42, LC/MS,Method C, (R_(t), m+1/z, purity), 0.99 min, 370, 75%.

(2R,2S)-[2-(2-aminoacetamido)]-3-(3-fluorophenyl)propanoic acid (43)

Method A, 17.7 mg (100%) of the trifluoroacetic acid salt of 43, LC/MS,Method C, (R_(t), m+1/z, purity), 0.36 min, 241, 100%.

(2R,2S)-[2-((S)-2-aminopropanamido)]-3-(3-fluorophenyl)propanoic acid(44)

Method A, 18.2 mg (99%) of the trifluoroacetic acid salt of 44, LC/MS,Method C, (R_(t), m+1/z, purity), 0.53 min, 255, 37% and 0.55 min, 255,58%.

(2R,2S)-[2-((S)-2-amino-3-(4-hydroxyphenyl)propanamido)]-3-(3-fluorophenyl)propanoicacid (45)

Method A, 19.0 mg (83%) of the trifluoroacetic acid salt of 45, LC/MS,Method C, (R_(t), m 1/z, purity), 0.73 min, 347, 42% and 0.74 min, 347,58%.

(2R,2S)-[2-((S)-2-amino-4-methylpentanamido)]-3-(3-fluorophenyl)propanoicacid (46)

Method A, 19.6 mg (96%) of the trifluoroacetic acid salt of 46, LC/MS,Method B, (R_(t), m+1/z, purity), 0.77 min, 297, 48% and 0.93 min, 297,41%.

(2R,2S)-[3-(4-fluorophenyl)]-2-((S)-pyrrolidine-2-carboxamido)propanoicacid (47)

Method A, 18.8 mg (95%) of the trifluoroacetic acid salt of 47, LC/MS,Method C, (R_(t), m 1/z, purity), 0.80 min, 281, 100%.

(2R,2S)-[3-(3-fluorophenyl)]-2-((S)-pyrrolidine-2-carboxamido)propanoicacid (48)

Method A, 17.3 mg (88%) of the trifluoroacetic acid salt of 48, LC/MS,Method B, (R_(t), m+1/z, purity), 0.54 min, 281, 45%, 0.66 min, 281,39%.

(R,S)-[2-((2R,3S)-2-amino-3-hydroxybutanamido)]-3-(4-fluorophenyl)propanoicacid (49)

Method A, 18.3 mg (92%) of the trifluoroacetic acid salt of 49, LC/MS,Method C, (R_(t), m+/z, purity), 0.39 min, 285, 44%, 0.52 min, 285, 56%.

(2R,2S)-[2-((2R,3S)-2-amino-3-hydroxybutanamido)]-3-(3-fluorophenyl)propanoicacid (50)

Method A, 18.4 mg (93%) of the trifluoroacetic acid salt of 50, LC/MS,Method C, (R_(t), m+1/z, purity), 0.39 min, 285, 44%, 0.52 min, 285,56%.

(2R,2S)-[2-((S)-2-amino-2-phenylacetamido)]-3-(4-fluorophenyl)propanoicacid (51)

Method A, 19.6 mg (91%) of the trifluoroacetic acid salt of 51, LC/MS,Method B, (R_(t), m+1/z, purity), 0.75 min, 317, 54%, 0.87 min, 317,46%.

(2R,2S)-[2-((S)-2-amino-2-phenylacetamido)]-3-(3-fluorophenyl)propanoicacid (52)

Method A, 19.9 mg (93%) of the trifluoroacetic acid salt of 52, LC/MS,Method B, (R_(t), m+1/z, purity), 0.75 min, 317, 54%, 0.87 min, 317,46%.

(2R,2S)-[2-((R)-2-aminopropanamido)]-3-(3-fluorophenyl)propanoic acid(53)

Method A, 18.2 mg (99%) of the trifluoroacetic acid salt of 53, LC/MS,Method C, (R_(t), m+1 z, purity), 0.54 min, 255, 100%.

(2R,2S)-[2-((S)-2-amino-3-phenylpropanamido)]-3-(3-fluorophenyl)propanoicacid (54)

Method A, 17.9 mg (81%) of the trifluoroacetic acid salt of 54, LC/MS,Method B, (R_(t), m+/z, purity), 0.80 min, 331, 55%, 0.98 min, 331, 45%.

(2R,2S)-[2-(2-((R,S)-amino-3-(4-fluorophenyl)propanamido)]-3-(4-fluorophenyl)propanoicacid (55)

Method A, 20.4 mg (89%) of the trifluoroacetic acid salt of 55, LC/MS,Method B, (R_(t), m+/z, purity), 0.85 min, 349, 42%, 1.01 min, 349, 58%.

(2R,2S)-[2-(2-((R,S)-amino-3-(4-fluorophenyl)propanamido)]-3-(3-fluorophenyl)propanoicacid (56)

Method A, 18.7 mg (81%) of the trifluoroacetic acid salt of 56, LC/MS,Method B, (R_(t), m 1/z, purity), 0.85 min, 349, 44%, 1.02 min, 349,56%.

(2R,2S)-[2-((S)-2-aminopropanamido)]-3-(3,4-fluorophenyl)propanoic acid(57)

Method A, 17.9 mg (93%) of the trifluoroacetic acid salt of 57, LC/MS,Method C, (R_(t), m+1 z, purity), 0.61 min, 273, 62%, 0.63 min, 273,30%.

(2R,2S)-[2-(2-aminoacetamido)]-3-(3,4-difluorophenyl)propanoic acid (58)

Method A, 16.5 mg (89%) of the trifluoroacetic acid salt of 58, LC/MS,Method C, (R_(t), m+1/z, purity), 0.48 min, 259, 100%.

(2R,2S)-[2-((S)-2-amino-3-(1H-indol-3-yl)propanamido)]-3-(2-fluorophenyl)propanoicacid (59)

Method A, 8.6 mg (36%) of the trifluoroacetic acid salt of 59, LC/MS,Method C, (R_(t), m+1 z, purity), 0.96 min, 370, 34%, 0.97 min, 370,53%.

(2R,2S)-[2-((S)-2-amino-3-(1H-indol-3-yl)propanamido)]-3-(3,4-difluorophenyl)propanoicacid (60)

Method A, 9.9 mg (40%) of the trifluoroacetic acid salt of 60, LC/MS,Method B, (R_(t), m+1/z, purity), 0.94 min, 388, 37%, 1.05 min, 388,48%.

(2R,2S)-[2-((S)-2-amino-3-(4-hydroxyphenyl)propanamido)]-3-(3,4-difluorophenyl)propanoicacid (61)

Method A, 22.6 mg (95%) of the trifluoroacetic acid salt of 61, LC/MS,Method B, (R_(t), m+1/z, purity), 0.72 min, 365, 51%, 0.85 min, 365,48%.

(2R,2S)-[2-((S)-2-amino-4-methylpentanamido)]-3-(3,4-difluorophenyl)propanoicacid (62)

Method A, 19.5 mg (91%) of the trifluoroacetic acid salt of 62, LC/MS,Method B, (R_(t), m 1 z, purity), 0.82 min, 315, 51%, 0.98 min, 315,42%.

(2R,2S)-[3-(2-fluorophenyl)-2-((S)-pyrrolidine-2-carboxamido)]propanoicacid (63)

Method A, 17.0 mg (86%) of the trifluoroacetic acid salt of 63, LC/MS,Method C, (R_(t), m+1/z, purity), 0.69 min, 281, 64%, 0.77 min, 281,31%.

(2R,2S)-[3-(3,4-difluorophenyl)-2-((S)-pyrrolidine-2-carboxamido)]propanoicacid (64)

Method A, 19.3 mg (94%) of the trifluoroacetic acid salt of 64, LC/MS,Method C, (R_(t), m+1 z, purity), 0.79 min, 299, 71%, 0.85 min, 299,29%.

(2R,2S)-[2-((2R,3S)-2-amino-3-hydroxybutanamido)]-3-(2-fluorophenyl)propanoicacid (65)

Method A, 18.1 mg (91%) of the trifluoroacetic acid salt of 65, LC/MS,Method C, (R_(t), m+1/z, purity), 0.35 min, 285, 32%, 0.48 min, 285,50%.

(2R,2S)-[2-((2R,3S)-2-amino-3-hydroxybutanamido)]-3-(3,4-difluorophenyl)propanoicacid (66)

Method A, 19.7 mg (95%) of the trifluoroacetic acid salt of 66, LC/MS,Method C, (R_(t), m+1/z, purity), 0.48 min, 303, 46%, 0.60 min, 303,48%.

(2R,2S)-[2-((S)-2-amino-2-phenylacetamido)]-3-(2-fluorophenyl)propanoicacid (67)

Method A, 19.5 mg (91%) of the trifluoroacetic acid salt of 67, LC/MS,Method B, (R_(t), m+1/z, purity), 0.73 min, 317, 52%, 0.84 min, 317,48%.

(2R,2S)-[2-((S)-2-amino-2-phenylacetamido)]-3-(3,4-difluorophenyl)propanoicacid (68)

Method A, 21.4 mg (96%) of the trifluoroacetic acid salt of 68, LC/MS,Method B, (R_(t), m+1/z, purity), 0.82 min, 335, 54%, 0.92 min, 335,46%.

(2R,2S)-[2-((R)-2-aminopropanamido)]-3-(3,4-difluorophenyl)propanoicacid (69)

Method A, 17.6 mg (91%) of the trifluoroacetic acid salt of 69, LC/MS,Method C, (R_(t), m+1/z, purity), 0.61 min, 273, 61%, 0.63 min, 273,30%.

(2R,2S)-[2-((S)-2-amino-3-phenylpropanamido)]-3-(3,4-difluorophenyl)propanoicacid (70)

Method A, 18.7 mg (81%) of the trifluoroacetic acid salt of 70, LC/MS,Method B, (R_(t), m+1/z, purity), 0.87 min, 349, 50%, 1.04 min, 349,50%.

(2R,2S)-[2-((R,S)-2-amino-3-(4-fluorophenyl)propanamido)-3-(2-fluorophenyl)propanoicacid (71)

Method A, 16.7 mg (72%) of the trifluoroacetic acid salt of 71, LC/MS,Method B, (R_(t), m+1/z, purity), 0.82 min, 349, 45%, 0.99 min, 349,55%.

(2R,2S)-[2-((R,S)-2-amino-3-(4-fluorophenyl)propanamido)-3-(3,4-difluorophenyl)propanoicacid (72)

Method A, 18.4 mg (77%) of the trifluoroacetic acid salt of 72, LC/MS,Method B, (R_(t), m+1/z, purity), 0.90 min, 367, 40%, 1.07 min, 367,56%.

(R,S)-[2-(2-amino-2-methylpropanamido)-3-(4-fluorophenyl)propanoic acid(73)

Method A, 13.4 mg (61%) of the trifluoroacetic acid salt of 73, LC/MS,Method B, (R_(t), m+1/z, purity), 0.65 min, 269, 87%.

(R,S)-[2-(2-amino-2-methylpropanamido)-3-(3-fluorophenyl)propanoic acid(74)

Method A, 12.4 mg (58%) of the trifluoroacetic acid salt of 74, LC/MS,Method B, (R_(t), m+/z, purity), 0.65 min, 269, 90%.

(R,S)-[2-((S)-2,6-diaminohexanamido)-3-(4-fluorophenyl)propanoic acid(75)

Method A, 17.7 mg (61%) of the ditrifluoroacetic acid salt of 75, LC/MS,Method C, (R_(t), m+/z, purity), 0.53 min, 312, 93%.

(R,S)-[2-((S)-2,6-diaminohexanamido)-3-(3-fluorophenyl)propanoic acid(76)

Method A, 18.1 mg (62%) of the ditrifluoroacetic acid salt of 76, LC/MS,Method C, (R_(t), m+1/z, purity), 0.54 min, 312, 92%.

(R,S)-[2-((S)-2-amino-2-(4-fluorophenyl)acetamido)-3-(4-fluorophenyl)propanoicacid (77)

Method A, 8.4 mg (28%) of the trifluoroacetic acid salt of 77, LC/MS,Method C, (R_(t), m+/z, purity), 0.91 min, 335, 41%, 1.01 min, 335, 38%.

(R,S)-[2-((2S,3S)-2-amino-3-methylpentanamido)-3-(4-fluorophenyl)propanoicacid (78)

Method A, 3.9 mg (17%) of the trifluoroacetic acid salt of 78, LC/MS,Method B, (R_(t), m+1/z, purity), 0.82 min, 297, 42%, 0.99 min, 297,47%.

(R,S)-[2-((2S,3S)-2-amino-3-methylpentanamido)-3-(3-fluorophenyl)propanoicacid (79)

Method A, 9.5 mg (34%) of the trifluoroacetic acid salt of 79, LC/MS,Method B, (R_(t), m+1/z, purity), 0.83 min, 297, 43%, 1.00 min, 297,47%.

(R,S)-2-((2S,3S)-2-amino-3-methylpentanamido)-3-(2-fluorophenyl)propanoicacid (80)

Method A, 16.1 mg (72%) of the trifluoroacetic acid salt of 80, LC/MS,Method B, (R_(t), m+1/z, purity), 0.80 min, 297, 44%, 0.97 min, 297,44%.

(R,S)-2-((2S,3S)-2-amino-3-methylpentanamido)-3-(3,4-difluorophenyl)propanoicacid (81)

Method A, 16.5 mg (69%) of the trifluoroacetic acid salt of 81, LC/MS,Method B, (R_(t), m+1/z, purity), 0.88 min, 315, 48%, 1.05 min, 315,47%.

(R,S)-2-((S)-2-amino-3-methylbutanamido)-3-(4-fluorophenyl)propanoicacid (82)

Method A, 15.0 mg (66%) of the trifluoroacetic acid salt of 82, LC/MS,Method B, (R_(t), m+1/z, purity), 0.70 min, 283, 47%, 0.89 min, 283,47%.

(R,S)-2-((S)-2-amino-3-methylbutanamido)-3-(3-fluorophenyl)propanoicacid (83)

Method A, 13.8 mg (63%) of the trifluoroacetic acid salt of 83, LC/MS,Method B, (R_(t), m+1/z, purity), 0.71 min, 283, 48%, 0.90 min, 283,48%.

(R,S)-2-((S)-2-amino-3-methylbutanamido)-3-(2-fluorophenyl)propanoicacid (84)

Method A, 16.2 mg (71%) of the trifluoroacetic acid salt of 84, LC/MS,Method B, (R_(t), m+1/z, purity), 0.68 min, 283, 51%, 0.88 min, 283,49%.

(R,S)-2-((S)-2-aminohexanamido)-3-(4-fluorophenyl)propanoic acid (85)

Method A, 15.6 mg (66%) of the trifluoroacetic acid salt of 85, LC/MS,Method B, (R_(t), m+1/z, purity), 0.87 min, 297, 46%, 1.03 min, 297,48%.

(R,S)-2-((S)-2-aminohexanamido)-3-(3-fluorophenyl)propanoic acid (86)

Method A, 14.5 mg (63%) of the trifluoroacetic acid salt of 86, LC/MS,Method B, (R_(t), m+1/z, purity), 0.88 min, 297, 46%, 1.03 min, 297,46%.

(R,S)-2-((S)-2-aminohexanamido)-3-(2-fluorophenyl)propanoic acid (87)

Method A, 16.0 mg (67%) of the trifluoroacetic acid salt of 87, LC/MS,Method B, (R_(t), m+1/z, purity), 0.85 min, 297, 45%, 1.00 min, 297,46%.

(R,S)-2-((S)-2-aminohexanamido)-3-(3,4-difluorophenyl)propanoic acid(88)

Method A, 16.6 mg (68%) of the trifluoroacetic acid salt of 88, LC/MS,Method B, (R_(t), m+1/z, purity), 094 min, 315, 44%, 1.08 min, 315, 44%.

(R,S)-2-((S)-2-amino-3-methylbutanamido)-3-(3,4-difluorophenyl)propanoicacid (89)

Method A, 17.1 mg (72%) of the trifluoroacetic acid salt of 89, LC/MS,Method B, (R_(t), m+1/z, purity), 0.77 min, 301, 51%, 0.95 min, 301,49%.

(R,S)-2-((S)-2-aminopentanamido)-3-(4-fluorophenyl)propanoic acid (90)

Method A, 13.2 mg (58%) of the trifluoroacetic acid salt of 90, LC/MS,Method B, (R_(t), m+1/z, purity), 0.75 min, 283, 45%, 0.92 min, 283,45%.

(R,S)-2-((S)-2-aminopentanamido)-3-(3-fluorophenyl)propanoic acid (91)

Method A, 13.1 mg (58%) of the trifluoroacetic acid salt of 91, LC/MS,Method B, (R_(t), m+1/z, purity), 0.76 min, 283, 49%, 0.92 min, 283,48%.

(R,S)-2-((S)-2-aminopentanamido)-3-(2-fluorophenyl)propanoic acid (92)

Method A, 14.7 mg (64%) of the trifluoroacetic acid salt of 92, LC/MS,Method B, (R_(t), m+1/z, purity), 0.72 min, 283, 46%, 0.89 min, 283,45%.

(R,S)-2-((S)-2-aminopentanamido)-3-(3,4-difluorophenyl)propanoic acid(93)

Method A, 15.8 mg (66%) of the trifluoroacetic acid salt of 93, LC/MS,Method B, (R_(t), m+/z, purity), 0.83 min, 301, 49%, 0.98 min, 301, 48%.

(R,S)-2-((S)-2-amino-4-(methylthio)butanamido)-3-(4-fluorophenyl)propanoicacid (94)

Method A, 6.9 mg (24%) of the trifluoroacetic acid salt of 94, LC/MS,Method B, (R_(t), m+/z, purity), 0.80 min, 315, 32%, 0.94 min, 315, 49%.

(R,S)-2-((S)-2-amino-4-(methylthio)butanamido)-3-(3-fluorophenyl)propanoicacid (95)

Method A, 7.2 mg (25%) of the trifluoroacetic acid salt of 95, LC/MS,Method B, (R_(t), m+1/z, purity), 0.80 min, 315, 33%, 0.94 min, 315,45%.

(R,S)-2-((S)-2-amino-4-(methylthio)butanamido)-3-(2-fluorophenyl)propanoicacid (96)

Method A, 9.7 mg (33%) of the trifluoroacetic acid salt of 96, LC/MS,Method B, (R_(t), m+1/z, purity), 0.78 min, 315, 40%, 0.91 min, 315,51%.

(R,S)-2-((S)-2-amino-4-(methylthio)butanamido)-3-(3,4-difluorophenyl)propanoicacid (97)

Method A, 8.4 mg (26%) of the trifluoroacetic acid salt of 97, LC/MS,Method B, (R_(t), m+1/z, purity), 0.87 min, 333, 39%, 1.00 min, 333,61%.

(R,S)-2-((S)-2-amino-3-hydroxypropanamido)-3-(4-fluorophenyl)propanoicacid (98)

Method A, trifluoroacetic acid salt of 98, LC/MS, Method B, (R_(t),m+1/z, purity), 0.46 min, 271, 35%, 0.56 min, 271, 30%.

(R,S)-2-((S)-2-amino-3-hydroxypropanamido)-3-(2-fluorophenyl)propanoicacid (99)

Method A, 15.6 mg (60%) of the trifluoroacetic acid salt of 99, LC/MS,Method B, (R_(t), m+1/z, purity), 0.41 min, 271, 42%, 0.51 min, 271,38%.

(R,S)-2-((S)-2-amino-3-hydroxypropanamido)-3-(3,4-difluorophenyl)propanoicacid (100)

Method A, 15.4 mg (54%) of the trifluoroacetic acid salt of 100, LC/MS,Method B, (R_(t), m+1/z, purity), 0.60 min, 289, 44%, 0.66 min, 289,33%.

(R,S)-3-(4-fluorophenyl)-2-(2-(methylamino)acetamido)propanoic acid(101)

Method A, 13.9 mg (68%) of the trifluoroacetic acid salt of 101, LC/MS,Method B, (R_(t), m+1/z, purity), 0.56 min, 255, 100%.

(R,S)-3-(3-fluorophenyl)-2-(2-(methylamino)acetamido)propanoic acid(102)

Method A, 13.6 mg (66%) of the trifluoroacetic acid salt of 102, LC/MS,Method B, (R_(t), m+/z, purity), 0.55 min, 255, 97%.

(R,S)-3-(2-fluorophenyl)-2-(2-(methylamino)acetamido)propanoic acid(103)

Method A, 12.0 mg (56%) of the trifluoroacetic acid salt of 103, LC/MS,Method B, (R_(t), m+1/z, purity), 0.52 min, 255, 96%.

(R,S)-3-(3,4-difluorophenyl)-2-(2-(methylamino)acetamido)propanoic acid(104)

Method A, 13.5 mg (62%) of the trifluoroacetic acid salt of 104, LC/MS,Method B, (R_(t), m+/z, purity), 0.66 min, 273, 91%.

(R,S)-2-((S)-2-amino-3-(1H-imidazol-4-yl)propanamido)-3-(4-fluorophenyl)propanoicacid (105)

Method A, 18.6 mg (82%) of the trifluoroacetic acid salt of 105, LC/MS,Method C, (R_(t), m+1/z, purity), 0.55 min, 321, 100%.

(R,S)-2-((S)-2-amino-3-(1H-imidazol-4-yl)propanamido)-3-(2-fluorophenyl)propanoicacid (106)

Method A, 20.5 mg (91%) of the trifluoroacetic acid salt of 106, LC/MS,Method C, (R_(t), m+1/z, purity), 0.50 min, 321, 100%.

(R,S)-2-((S)-2-amino-3-(1H-imidazol-4-yl)propanamido)-3-(3,4-difluorophenyl)propanoicacid (107)

Method A, 20.7 mg (91%) of the trifluoroacetic acid salt of 107, LC/MS,Method C, (R_(t), m+1/z, purity), 0.64 min, 339, 85%.

(2R,2S)-[((2S,3R)-2-amino-3-hydroxybutanamido)]-3-(4-fluorophenyl)propanoicacid (108)

Method A, 13.2 mg (63%) of the trifluoroacetic acid salt of 108, LC/MS,Method A, (R_(t), m+1/z, Purity), 1.93 min, 285, 36%, 4.24 min, 285,32%.

(S)-2-((S)-2-amino-4-methylpentanamido)-3-(3-fluorophenyl)propanoic acid(46a) and(R)-2-((S)-2-amino-4-methylpentanamido)-3-(3-fluorophenyl)propanoic acid(46b)

Method A, 18.4 mg of the trifluoroacetic acid salts of 46a and 46b;LC/MS, Method C (R_(t), m+1/z, purity) 0.96 min, 297, 95%). Thediastereomers were separated by chromatography on silica gel (500 mg)using 36:2:1 isopropanol:methanol:concentrated ammonium hydroxide as themobile phase to give 2.7 mg of 46a as the higher R_(f) diastereomers and3.0 mg of 46b as the lower R_(f) diastereomer. ¹HNMR (CD₃OD) 46a δ 0.97(d, 3H, J=5.8 Hz), 0.97 (d, 3H, J=5.8 Hz), 1.64 (m, 3H), 3.08 (dd, 1H,J=14.0 and 8.3 Hz), 3.26 (dd, 1H, J=13.9 and 4.8 Hz), 3.76 (t, 1H, J=8.0Hz), 4.47 (dd, 1H, J=8.3 and 4.9), 6.90 (dt, 1H, J=8.4 and 2.1 Hz), 7.02(d, 1H, J=8.0 Hz), 7.03 (d, 1H, J=7.6 Hz), and 7.08 (dd, 1H, J=7.9 and6.2 Hz); 46b δ 0.80 (d, 3H, J=6.6 Hz), 0.82 (d, 3H, J=6.5 Hz), 1.07 (m,1H), 1.31 (m, 1H), 1.51 (m, 1H), 2.82 (dd, 1H, J=14.2 and 11.0 Hz), 3.42(dd, 1H, J=14.2 and 3.9 Hz), 3.67 (t, 1H, J=7.3 Hz), 4.62 (dd, 1H,J=11.0 and 4.0 Hz), 6.90 (dt, 1H, J=8.5 and 2.5 Hz), 6.92 (d, 1H, J=10.1Hz), 6.99 (d, 1H, J=7.7 Hz), and 7.08 (dd, 1H, J=8.0 and 6.1 Hz).

(S)-2-((S)-2-amino-3-(4-hydroxyphenyl)propanamido)-3-(3-fluorophenyl)propanoicacid (45a) and(R)-2-((S)-2-amino-3-(4-hydroxyphenyl)propanamido)-3-(3-fluorophenyl)propanoicacid (45b)

Method A, 17.0 mg of the trifluoroacetic acid salts of 45a and 45b;LC/MS, Method C(R_(t), m+1/z, purity) 0.74 min, 347, 97%. Thediastereomers were separated by chromatography on silica gel (500 mg)using 36:2:1 isopropanol:methanol:concentrated ammonium hydroxide as themobile phase to give 1.0 mg of 45b as the lower R_(f) diastereomer. Thehigher R_(f) diastereomer 45a resulting from this chromatography wascontaminated with the lower R_(f) diastereomer 45b. The higher R_(f)diastereomer was separated by chromatography on silica gel (500 mg)using 18:2:1 isopropanol:methanol:concentrated ammonium hydroxide as themobile phase to give 1.1 mg of 45a as the higher R_(f) diastereomer.¹HNMR (CD₃OD) 45a δ 2.83 (br s, 1H), 3.07 (br m, 1H), 3.15 (br s, 1H),3.22-3.28 (br m, 1H), 3.83 (br s, 1H), 4.53 (br s, 1H), 6.77 (d, 1H,J=7.7 Hz), 6.91 (t, 1H, J=8.4 Hz), 7.02 (d, 1H, J=10.1 Hz), 7.06 (d, 1H,J=7.5 Hz), 7.10 (d, 2H, J=7.8 Hz), 7.25 (m, 1H); 45b δ 2.71 (dd, 1H,J=14.1 and 7.7 Hz), 2.91 (m, 2H), 3.20 (dd, 1H, J=13.8 and 3.8 Hz), 3.94(bt, 1H, J=13.6 Hz), 4.57 (dd, 1H, J=9.0 and 4.3 Hz), 6.72 (d, 2H, J=8.5Hz), 6.95 (m, 5H), 7.24 (m, 1H).

Semi-preparative chromatography was performed on an Agilent ZorbaxEclipse XDB C18 5-micron, 9.4×250 mm column. Mobile phases were mixturesof 1:1 MeOH:MeCN w/5 mM NH₄OAc and 5 mM NH₄OAc in Milli-Q water. Elutionwas performed at 2 mL/min using a programmed gradient with detection at220 nm. Real time detection was monitored using a chart recorder. Whennecessary, fractions were analyzed by analytical LCMS. Solutions wereevaporated to residues and then placed under vacuum. Only partialremoval of ammonium acetate was accomplished.

(S)-2-((S)-2-aminopropanamido)-3-(4-fluorophenyl)propanoic acid (25a)and (R)-2-((S)-2-aminopropanamido)-3-(4-fluorophenyl)propanoic acid(25b)

16.0 mg of a crude sample of the trifluoroacetic acid salts of 25a and25b was chromatographed on a 500-mg column of silica gel using mixturesof isopropanol-methanol-concentrated ammonium hydroxide to obtain 4.5 mgof a mixture of 25a and 25b free of higher and lower Rf contaminants. Aportion (1.8 mg) was dissolved in 1.0 mL of 85/15 1:1 MeOH:MeCN w/5 mMNH₄OAc and 5 mM NH₄OAc in Milli-Q water and the solution was injected onto the column. The stereoisomers were eluted using a gradient program of15-100% 1:1 MeOH:MeCN w/5 mM NH₄OAc over 30 minutes. Fractionscontaining the completely separated isomers were evaporated to dryresidues which were placed in a vacuum oven at 40° C. for 24 hours togive 2.0 mg of 25a as the earlier retention time isomer (10.8-13.2 min),¹HNMR (D₂O) 25a δ 1.44 (d, 3H, J=7.1 Hz), 2.93 (dd, 1H, J=14.0 and 8.6Hz), 3.12 (dd, 1H, J=14.1 and 5.5 Hz), 3.93 (br q, 1H, J=6.6 Hz), 4.37(dd, 1H, J=8.6 and 5.5 Hz), 7.04 (t, 2H, J=8.9 Hz), 7.22 (m, 2H) and 1.0mg of 25b as the later retention time isomer (14.4-15.6 min), ¹HNMR(D₂O) 25b δ 1.17 (d, 3H, J=6.5 Hz), 2.83 (dd, 1H, J=14.1 and 9.8 Hz),3.21 (dd, 1H, J=14.2 and 4.7 Hz), 3.89 (br s, 1H), 4.45 (dd, 1H, J=9.7and 4.7 Hz), 7.03 (t, 2H, J=8.9 Hz), 7.21 (m, 2H).

(S)-2-((S)-2-aminopropanamido)-3-(3-fluorophenyl)propanoic acid (44a)and (R)-2-((S)-2-aminopropanamido)-3-(3-fluorophenyl)propanoic acid(44b)

15.6 mg of a crude sample of the trifluoroacetic acid salts of 44a and44b were chromatographed on a 500-mg column of silica gel using mixturesof isopropanol-methanol-concentrated ammonium hydroxide to obtain amixture of 44a and 44b free of higher and lower Rf contaminants.Approximately 2.0 mg was dissolved in 3 mL of 90/10 1:1 MeOH:MeCN w/5 mMNH₄OAc and 5 mM NH₄OAc in Milli-Q water. Three 1.0-mL injections on tothe column were made eluting the stereoisomers using a gradient programof 10-100% 1:1 MeOH:MeCN w/5 mM NH₄OAc over 60 minutes. Fractionscontaining the completely separated isomers were evaporated to dryresidues to give 1.2 mg of 44a as the earlier retention time isomer(13.2-15.6 min), ¹HNMR (D₂O) 44a δ 1.42 (d, 3H, J=7.0 Hz), 2.95 (dd, 1H,J=14.0 and 8.7 Hz), 3.16 (dd, 1H, J=14.0 and 5.3 Hz), 3.89 (br s, 1H),4.40 (dd, 1H, J=8.7 and 5.3 Hz), 6.96-7.02 (m, 2H), 7.04 (d, 1H, J=7.7Hz), 7.31 (m, 1H) and 1.2 mg of 44b as the later retention time isomer(16.8-19.6 min), ¹HNMR (D₂O) 44b δ 1.19 (d, 3H, J=7.0 Hz), 2.86 (dd, 1H,J=14.1 and 10.0 Hz), 3.26 (dd, 1H, J=14.1 and 4.7 Hz), 3.94 (br q, 1H,J=6.7 Hz), 4.49 (dd, 1H, J=10.0 and 4.8 Hz), 6.96-7.00 (m, 2H), 7.04 (d,1H, J=7.7 Hz), 7.30 (m, 1H).

(S)-2-((2S,3R)-2-amino-3-hydroxybutanamido)-3-(4-fluorophenyl)propanoicacid (108a) and(R)-2-((2S,3R)-2-amino-3-hydroxybutanamido)-3-(4-fluorophenyl)propanoicacid (108b)

13.2 mg of a crude sample of the trifluoroacetic acid salts of 108a and108b were chromatographed on a 500-mg column of silica gel usingmixtures of isopropanol-methanol-concentrated ammonium hydroxide toobtain 2.0 mg of a mixture of 108a and 108b free of higher and lower Rfcontaminants. Three 1.0-mL injections on to the column were made elutingthe stereoisomers using a gradient program of 90/10 1:1 MeOH:MeCN w/5 mMNH₄OAc and 5 mM NH₄OAc in Milli-Q water for 1.0 min followed by 10-100%1:1 MeOH:MeCN w/5 mM NH₄OAc over 60 minutes. Fractions containing thecompletely separated isomers were evaporated to dry residues to give 0.8mg of 108a as the earlier retention time isomer (12.8-17.2 min), ¹HNMR(D₂O) 108a δ 1.25 (d, 3H, J=6.5 Hz), 2.97 (dd, 1H, J=13.9 and 8.2 Hz),3.15 (dd, 1H, J=14.2 and 5.6 Hz), 3.78 (br d, 1H, J=5.2 Hz), 4.13(quintet, 1H, J=6.2 Hz), 4.44 (dd, 1H, J=8.4 and 5.6 Hz), 7.07 (t, 2H,J=8.9 Hz), 7.26 (m, 2H) and 108b as the later retention time isomer(17.6-23.2 min), ¹HNMR (D₂O) 108b δ 0.86 (d, 3H, J=6.2 Hz), 2.88 (dd,1H, J=14.3 and 10.2 Hz), 3.26 (dd, 1H, J=14.3 and 4.6 Hz), 3.64 (br s,1H), 3.81 (br quintet, 1H, J=6.3 Hz), 4.51 (dd, 1H, J=10.2 and 4.6 Hz),7.07 (t, 2H, J=8.9 Hz), 7.28 (m, 2H).

(S)-2-((S)-2-amino-2-phenylacetamido)-3-(3-fluorophenyl)propanoic acid(52a) and(R)-2-((S)-2-amino-2-phenylacetamido)-3-(3-fluorophenyl)propanoic acid(52b)

17.0 mg of a crude sample of the trifluoroacetic acid salts of 52a and52b were chromatographed on a 500-mg column of silica gel using mixturesof isopropanol-methanol-concentrated ammonium hydroxide to obtain amixture of 52a and 52b. This material was then triturated under coldmethanol to afford 4.3 mg of 52a and 52b as a white solid. Approximately4.0 mg was dissolved in 6 mL of 90/10 1:1 MeOH:MeCN w/5 mM NH₄OAc and 5mM NH₄OAc in Milli-Q water. Six injections of 1.0 mL were made and theisomers were eluted using a gradient program of 90/10 1:1 MeOH:MeCN w/5mM NH₄OAc and 5 mM NH₄OAc in Milli-Q water for 1.0 min followed by10-100% 1:1 MeOH:MeCN w/5 mM NH₄OAc over 60 minutes. Fractionscontaining the completely separated isomers were evaporated to dryresidues to give 2.0 mg of 52a as the earlier retention time isomer(24.8-27.6 min), ¹HNMR (CD₃OD) 52a δ 3.06 (br m, 1H), 3.24 (br m, 1H),3.94 (br s, 1H) 4.46 (br s, 1H), 6.87 (t, 1H, J=8.5 Hz), 6.90-6.99 (m,2H), 7.18 (m, 1H), 7.23-7.40 (m, 5H) and 1.4 mg of 52b as the laterretention time isomer (27.6-29.6 min), ¹HNMR (CD₃OD) 52b δ 2.85 (dd, 1H,J=13.7 and 8.2 Hz), 3.15 (br md, 1H), 3.95 (br s, 1H), 4.55 (br s, 1H),6.64-6.71 (m, 2H), 6.76 (t, 1H, J=8.2 Hz), 7.00 (m, 1H), 7.24-7.39 (m,5H).

(S)-2-((S)-2-aminopropanamido)-3-(2-fluorophenyl)propanoic acid (36a)and (R)-2-((S)-2-aminopropanamido)-3-(2-fluorophenyl)propanoic acid(36b)

10.4 mg of the mixture of the diastereomers 36a and 36b were separatedby chromatography on silica gel (500 mg) using 36:2:1isopropanol:methanol:concentrated ammonium hydroxide as the mobile phaseto give 1.5 mg of 36a as the higher R_(f) diastereomer and 1.9 mg of 36bas the lower R_(f) diastereomer. ¹HNMR (D₂O) 36a δ 1.46 (d, 3H, J=7.1Hz), 3.01 (dd, 1H, J=13.9 and 8.6 Hz), 3.25 (dd, 1H, J=13.8 and 5.4 Hz),3.96 (br s, 1H), 4.46 (dd, 1H, J=8.5 and 5.6 Hz), 7.09-7.16 (m, 2H),7.26-7.32 (m, 2H). ¹HNMR (D₂O) 36b δ 1.21 (d, 3H, J=7.1 Hz), 2.93 (dd,1H, J=14.1 and 10.0 Hz), 3.32 (dd, 1H, J=14.0 and 4.8 Hz), 3.96 (br q,1H, J=6.6 Hz), 4.58 (dd, 1H, J=9.9 and 4.9 Hz), 7.08-7.16 (m, 2H),7.26-7.31 (m, 2H).

Referring now to FIGS. 14-22, IC₅₀ values have been determined using thefollowing procedures: Day 1: an overnight culture was set up:Pseudomonas aeruginosa (PA14) was inoculated in 5 mL of lysogeny broth(LB) and shaken at 180 rpm at 37° C. for 18-24 hours. Day 2: 90 μL ofPA14 diluted 1:100 in completed M63 media was added into the test wellsof a 96 well plate. Next, 10 μL of the active compound at varyingconcentrations was added to the wells creating a concentration gradientgoing down the plate; the bacterial control substituted 10 μL of 10%DMSO in place of active compound. The media control wells contain onlyM63 and 10% DMSO. The plate was incubated at 37° C. for 18-24 hours. Day3: inoculum was removed from the plate and each well was washed twicewith 100 μL of DI H₂O; the DI water was removed after each wash. Next,125 μL of 0.1% Crystal Violet (CV) was added to each well. Wells wereallowed to stain for 10 min. CV was removed and each well was washedthree additional times with 200 μL of DI water. The plate was allowed todry completely. Day 4: 150 μL of 30% acetic acid was added to each welland CV was allowed to completely dissolve for 15 min. (FIG. 26). Opticaldensity (OD) of each well was measured at 550 nm, blanking to mediacontrol wells.

FIG. 23 shows representative graphs for a sample compound that were usedto determine corresponding IC₅₀ and R² values. For example, each testwell is compared to the bacterial control wells in order to obtain apercent control. These values are first plotted on a bar graph with they-axis as percent control and x-axis as the well concentration after theactive compound was added. If an IC₅₀ (50% bacteria growth) is shown,that concentration is determined to be the IC₅₀ value. If a clear IC₅₀cannot be determined from bar graph, but there is a clear trend, ascatter plot of the data is graphed and a trend line is generated. Ifthe r² value of the equation for the trend line is greater than 0.97, itis used to generate the IC₅₀ value.

Certain dipeptides containing fluorophenylalanines (“FPhe”) at eitherthe C- or N-terminus can be more active by virtue of increased uptake bythe bacteria followed by hydrolysis to the active FPhe. These dipeptidescould also be more selective by virtue of either less uptake bymammalian cells or lack of a mammalian enzyme capable hydrolyzing thedipeptide back to FPhe. Therefore, a procedure was designed to makeunnatural dipeptides and screen for their effects.

Unnatural dipeptides of generic formula II (below) were made based onearlier work showing potent activity of the precursorfluorophenylalanines I (below) against P. aeruginosa (Pa). These simplefluorinated phenylalanine analogs are reported to be anti-metabolites, asearch was initiated for selective and potent prodrug derivatives of I,made by incorporating its isomers into unnatural dipeptides II and III(below). L-Ala-(R,S)-4-fluorophenylalanine andL-Ala-(R,S)-2-fluorophenylalanine were early products derived from thisapproach.

For I, II, and III: —F=2-F, 3-F, 4-F, or 3,4-diF

Dipeptides containing fluorophenylalanines (“FPhe”) disclosed hereinwere screened for antimicrobial activity using Community for OpenAntimicrobial Drug Discovery (CO-ADD) program. The screening resultsprovided that the unnatural dipeptides includingL-Ala-(R,S)-4-fluorophenylalanine and L-Ala-(R,S)-2-fluorophenylalanineexhibited inhibitory effects on the growth of Cryptococcus neoformans(Cn). In addition, these dipeptides exhibited potent ability to inhibitthe growth of P. aeruginosa (Pa) in a phenylalanine deficient biofilmassay.

A comprehensive structure-activity relationship (SAR) using the resultsderived from L-Ala-(R,S)-4-fluorophenylalanine andL-Ala-(R,S)-2-fluorophenylalanine was performed. The initial focus wason synthesizing analogs of generic structure II (above) in which theN-terminal amino acid would be a natural amino acid and the C-terminalamino acid a racemic fluorinated phenylalanine I (above).

Exemplary lots of the dipeptides above exhibited a minimum inhibitoryconcentration (MIC) against Cn of 8 μg/mL. Structures and results fromthe screens are summarized below.

4-Fluorophenylalanine derivative

2-Fluorophenylalanine derivative

PS = Preliminary screen HC = Hit confirmation Cn = Cryptococcusneoformans Tox = Toxic aagainst Hk cell line Pa = Pseudomonas aeruginosa

Referring now to FIG. 30, activity against Ca and/or Cn was confirmedfor three regioisomers of L-Ala-(R,S)-FPhe (e.g., compounds 25, 36, and44), and preliminary activity for compounds 30, 43, 45, 46, 48, and 52are submitted for screening. All of these results were obtained on tworeplicated lots of product as mixtures of enantiomers or diastereomers.In addition individual diastereomers of a seventh compound (108) wereprepared for screening since the compound 108 had shown activity againstPa in a biofilm assay.

Referring now to FIG. 31, the individual diastereomers were separated,characterized, and submitted for screening using CO-ADD program. Theresults from both the preliminary screen (PS) and subsequent hitconfirmation (HC) are shown in FIG. 32. The separation methods are shownbelow.

Referring to FIG. 32, with the exception of one case (e.g.,L-Ala-(R,S)-2-fluorophenylalanine, 36a and 36b), preliminary screeningindicated that activity against Cn and/or Ca can be observed when usinga single diastereomer. This result together with regioisomerdiscrimination between Cn and/or Ca can be a good indication that aselective molecular mechanism of action exists. The (S) stereochemistrywas tentatively assigned to the fluorinated phenylalanine in the activedipeptides as some of the unnatural monopeptides tested exhibitedactivity against Cn and/or Ca only in the (S) isomer (data not shown).All preliminary activity was confirmed in the hit confirmation assay(HC), except for compounds 36a, 46a, and 52a.

None of the hit confirmed compounds showed toxicity in the humanembryonic kidney cell (Hk) or haemolysis (Hm) assay. Further, hitconfirmation was observed in a new area—Acinetobacter baumannii—forcompounds 25a, 44a, and 45a. This activity was not seen in thepreliminary screen.

TABLE 1 Abbreviation Name Description Strain Organsim Type Media SaStaphylococcus aureus MRSA ATCC 43300 Bacteria G+ve CAMHB Ec Escherichiacoli FDA control ATCC 25922 Bacteria G−ve CAMHB Kp Klebsiella pneumoniaeMDR ATCC 700603 Bacteria G−ve CAMHB Ab Acinetobacter baumannii Typestrain ATCC 19606 Bacteria G−ve CAMHB Pa Pseudomonas aeruginosa Typestrain ATCC 27853 Bacteria G−ve CAMHB Ca Candida albicans CLSI referenceATCC 90028 Fungi Yeast YNB Cn Cryptococcus neoformans var. Type strainH99; ATCC Fungi Yeast YNB grubii 208821 Hk Human embryonic kidney cellsHEK-293 ATCC CRL-1573 Human Eukaryotes DMEM 10% FBS Hm Human red bloodcells RBC Human Eukaryotes

The minimum inhibitory concentration (MIC) was determined following theCLSI (Clinical Laboratory and Standards Institute) guidelines,identifying the lowest concentration at which full inhibition of thebacteria or fungi has been detected. Full inhibition of growth has beendefined at ≤20% growth (or >80% inhibition), and concentrations haveonly been selected if the next highest concentration displayed fullinhibition (i.e. 80-100%) as well (eliminating ‘singlet’ activeconcentration). Note that MIC values are discrete values based on theconcentration in a specific well. Any value with > indicates that sampledisplays no activity (low DMax value) or partial activity at the highesttested concentration (higher DMax value).

CC₅₀ (Concentration at 50% Cytotoxicity) were calculated by curvefitting the inhibition values vs. log (concentration) using Sigmoidaldose-response function, with variable values for bottom, top and slope.The curve fitting is implemented using Pipeline Pilot's dose-responsecomponent (giving similar results to similar tools such as GraphPad'sPrism and IDBS's XlFit). Any value with > indicates a sample with noactivity (low DMax value) or samples with CC₅₀ values above the maximumtested concentration (higher DMax value).

HC₁₀ (Concentration at 10% Haemolytic activity) were calculated by curvefitting the inhibition values vs. log (concentration) using Sigmoidaldose-response function, with variable values for bottom, top and slope.The curve fitting is implemented using Pipeline Pilot's dose-responsecomponent (giving similar results to similar tools such as GraphPad'sPrism and IDBS's XlFit). The curve fitting resulted in HC₅₀ (50%)values, which are converted into HC₁₀ by HC₁₀=HC₅₀*(10/90){circumflexover ( )}(1/Slope); Any value with > indicates a sample with no activity(low DMax value) or samples with HC₁₀ values above the maximum testedconcentration (higher DMax value).

DMax (Maximum Response) represents the highest percentage inhibitionresponse for all concentrations tested. The value helps to indicate ifsamples are displaying only partial response at the screenedconcentration, suggesting that the sample might be fully active at ahigher concentration or if the sample only exhibits partial inhibition.In addition, the value indicates if samples have been active but curvefitting failed, mostly due to the fact that only the single highestconcentration was active.

For quality control, all screening is performed as two replica (n=2),with both replicas on different assay plates, but from single platingand performed in a single screening experiment (microbial incubation).Each individual value is reported in the table. In addition, two valuesare used as quality controls for individual plates:Z′-Factor=[1−(3*(sd(NegCtrl)+sd(PosCtrl))/(average(PosCtrl)−average(NegCtrl)))]and Standard Antibiotic controls at different concentrations (>MIC and<MIC). The plate passes the quality control if Z′-Factor>0.4 andStandards are active and inactive at highest and lowest concentrations,respectively (this data is not supplied by CO-ADD).

Selection of Hits: Samples with MIC (≤16 μg/mL or ≤10 μM) are declaredas a hit. For toxicity (cytotoxicity and haemolytic activity) allsample/s with CC₅₀/HC₁₀≤maximum tested concentration are consideredactive, or toxic. Since the maximum tested concentration is the same fortoxicity and antimicrobial activity, no therapeutic index (MIC/CC₅₀ orMIC/HC₁₀) can be calculated and all sample/s with toxic activity areflagged. Inactive samples are those with MIC/CC₅₀/HC₁₀>16 μg/mL or >10μM.

The term, “Hit” in FIG. 51 indicates the number of organism-classes (GN,GP and FG) the compound has been found active against, 0=no activity.

The term, “Tox” in FIG. 51 indicates samples with cytotoxicity(Dmax>50%) or haemolytic activity (Dmax>10%). This more stringentthreshold is used as the samples were tested at the same concentrationrange as MIC, but Toxicity assays are usually run at higher sampleconcentration to evaluate their therapeutic index (difference betweenMIC and CC₅₀). This stringent threshold is used to flag any partialtoxicity, which would show well defined toxicity (CC₅₀ or HC₁₀) athigher concentrations.

The term, “Act” in FIGS. 52-53 indicates the number of organism-classes(GN, GP and FG) the compound has been found active against, 0=noactivity.

The term, “Sel” in FIGS. 52-53 indicates compounds that have beenselected for further dose response studies, Hit-Confirmation. Theselection includes all active as well as compounds with ambiguousresults requiring confirmation of activity or inactivity.

FIGS. 51-53 show data from screenings performed by CO-ADD. See Table 1,above, for abbreviations and conditions used in FIGS. 51-53. FIG. 51shows MIC, CC₅₀ (cytotoxicity), and HC₁₀ (haemolytic activity) valuesfor each organism, as well as DMax (maximum response) values. For MIC,cytotoxicity, and haemolysis, individual data points for import inin-house databases. Haemolysis also reports the HC₅₀ values, which isused to calculate the HC₁₀ values. Compounds were plated as a 2-folddose response from 32 to 0.25 μg/mL (or 20 to 0.156 μM), with a maximumof 0.5% DMSO, final in assay concentration.

Referring now to FIGS. 52 and 53, inhibition of bacterial growth wasdetermined measuring absorbance at 600 nm (OD600), using a Tecan M1000Pro monochromator plate reader. The percentage of growth inhibition wascalculated for each well, using the negative control (media only) andpositive control (bacteria without inhibitors) on the same plate asreferences.

Growth inhibition of C. albicans was determined measuring absorbance at530 nm (OD530), while the growth inhibition of C. neoformans wasdetermined measuring the difference in absorbance between 600 and 570 nm(OD600-570), after the addition of resazurin (0.001% finalconcentration) and incubation at 35° C. for additional 2 h. Theabsorbance was measured using a Biotek Synergy HTX plate reader. Thepercentage of growth inhibition was calculated for each well, using thenegative control (media only) and positive control (bacteria withoutinhibitors) on the same plate as references. Percentage growthinhibition of an individual sample is calculated based on Negativecontrols (media only) and Positive Controls (bacterial/fungal mediawithout inhibitors). Negative inhibition values indicate that the growthrate (or OD600) is higher compared to the Negative Control(Bacteria/fungi only, set to 0% inhibition). The growth rates for allbacteria and fungi has a variation of −/+10%, which is within thereported normal distribution of bacterial/fungal growth.

Any significant variation (or outliers/hits) is identified by themodified Z-Score, and actives are selected by a combination ofinhibition value and Z-Score. Z-Score analysis is done to investigateoutliers or hits among the samples. The Z-Score is calculated based onthe sample population using a modified Z-Score method which accounts forpossible skewed sample population. The modified method uses median andMAD (median average derviation) instead of average and sd, and a scalingfactor (see: Iglewicz, B. & Hoaglin, D. C. Volume 16: How to Detect andHandle Outliers. The ASQC Basic Reference in Quality Control:Statistical Techniques, 1993): M(i)=0.6745*(x(i)−median(x))/MAD). M(i)values of >|2.5| (absolute) label outliers or hits. For quality control,all screening is performed as two replica (n=2), with both replicas ondifferent assay plates, but from single plating and performed in asingle screening experiment (microbial incubation).

Each individual value is reported in FIGS. 51-53. In addition, twovalues are used as quality controls for individual plates: Z′-Factor[1−(3*(sd(NegCtrl)+sd(PosCtrl))/(average(PosCtrl)−average(NegCtrl)))]and Standard Antibiotic controls at different concentrations (>MIC and<MIC). The plate passes the quality control if Z′-Factor>0.4 andStandards are active and inactive at highest and lowest concentrations,respectively. This data is not supplied by CO-ADD. Active samples aredefined as those with inhibition values equal to or above 80% andabs(Z-Score) above 12.51 for either replicate (n=2 on different plates)were classed as active. Partially active compounds are defined as thosewith inhibition values between 50.9%-79.9% or abs(Z-Score) below 12.51.Inactive compounds with inhibition values below 50% and/or abs(Z-Score)below 12.51.

Materials and Methods

According to Curran and Wipf (Chemical & Engineering News, p. 7, Mar.17, 1997), “[c]ombinatorial synthesis is the intentional construction ofa collection of molecules based on logical design and involving theselective combination of building blocks by means of simultaneouschemical reactions. The collection of molecules resulting from acombinatorial synthesis is a combinatorial library.”

The following is an example of the general lab procedure used to makeacylated unnatural amino acids using simple, inexpensive equipment andsolid-phase combinatorial chemistry procedures. Referring now to FIG.33, six diastereomeric unnatural dipeptide products were synthesized.Each reaction product contains a mixture of the two diastereomers I andII: with (R)- or (S)-4-fluorophenylalanine at the N-terminus, and amixture of (S)- and (R)-fluorinated phenylalanines at the C-terminus.

Referring now to Scheme 7, solid-phase chemistry and alkylating agentsR¹—X and Boc-protected 4-F Phenylalanine are employed for the synthesisof a small combinatorial library of unnatural dipeptides 109 using5-step syntheses on a 50 μmol scale. The five-step synthetic processinvolves introduction of two variables; R¹—X and (R)- or(S)—N-Boc-protected 4-fluorophenylalanine onto the activated glycineamino acid scaffold 110. Briefly, R¹ is present as the side chain inresin-bound protected amino acids 111. After neutralization to 112, thesecond group is introduced by forming an amide bond by an amineacylation (catalyzed with HOBt and DIC) with Boc-protected (R)- or(S)-4-fluorophenylalanine. After reaction for several days, followed bythorough washing, the ester link of the product to the resin 113 iscleaved with a strong acid (trifluoroacetic acid). The Boc protectinggroup is simultaneously removed. The resulting target molecule 109 is insolution and is separated from spent resin by filtration.

In each instance, one position of the simple “Bill-Board” equipment isused to carry out this multiple solid-phase reaction. A representativeBill-Board grid is shown in FIG. 34. This method can be used tosynthesize the exemplary molecules shown in FIG. 35.

Following completion of the reaction sequence, 50 μmols of startingmaterial bound to resin leads to the isolation of approximately 10-20 mgof each crude product. Following purification, NMR, LC/MS, and UV dataare collected. Samples of the molecules made are tested for theirability to inhibit the growth of P. aeruginosa, and are also besubmitted for testing in Australia by the Community for OpenAntimicrobial Drug Discovery (CO-ADD). They will assess their ability toinhibit the growth of a panel of antibiotic resistant microbes.

Referring now to Scheme 6, 50 μmols of 110, an imine-activated aminoacid bound by a Wang linker to polystyrene beads (known as a “Wangresin”) is added by pipetting equal volumes of an isopycnic (neutralbuoyancy) suspension of the resin to each of the vessels. All caps ofthe Bill-Board vessel are removed, the resin is washed 3 times withapproximately 2-mL portions of NMP (N-methyl pyrrolidinone), and thebottom cap of the reaction vessel is replaced. 0.5 mL of a 0.20 Msolution of the t-butylimino-tri(pyrrolidino)phosphorane (BTPP) in NMP(100 μmols, 2 equiv) is added, followed by 0.5 mL of the 0.20 M R¹—Xsolution in NMP (100 μmols, 2 equiv). The vessel is capped, inverted tomix the contents, and left at room temperature for 5 days. The remainingreagents are drained, and the alkylated resin product 111 is filteredand washed with 3 mL of tetrahydrofuran (THF). New caps are obtained forthe reaction vessel, and the bottom cap applied. Approximately 2.5 mL of1N aqueous hydrochloric acid/tetrahydrofuran mixture (1:2) is added. Thevessel is capped, inverted a few times to mix the contents, then left atroom temperature for 20 minutes. The resultant hydrolyzed resin-boundproduct (112a) is filtered and washed one time with 3 mL of THF, then2×2.5 mL×3 minutes with 0.20 M diisopropylethylamine (DIEA) in NMP, andthen 2×2.5 mL NMP. After capping the bottom of the reaction vessel witha clean cap, 1.0 mL of the acylating agent, 0.25 M R²—CO₂H in 0.25 Mhydroxybenzotriazole (HOBt) in NMP (both 250 μmol, 5 equiv) are added tothe deprotected resin 112b in the vessel. 0.5 mL of a 0.50 M solution ofdiisopropylcarbodiimide (“DIC”, 250 μmol, 5 equiv) in NMP, is added tothe vessel. The top of the reaction vessel is capped and inverted, andleft at room temperature for 2 days. The resultant acylated resinproduct is filtered and washed (114) two times with 2 mL of NMP, twotimes with 2 mL of THF and six times with 2 mL of dichloromethane(CH₂Cl₂). 2 mL of trifluoroacetic acid (TFA)-CH₂Cl₂—H₂O (35:60:5) isadded to the vessel, after which the reaction is allowed to stand for 30min without agitation. The resin is rinsed once with 2 mL ofTFA/CH₂Cl₂/H₂O. 100 μL (0.1 mL) of the product is then transferred to anHPLC autosampler vial for purification, and the remainder of the crudeproduct is analyzed by TLC (18:2:1 isopropanol:methanol:ammoniumhydroxide) and visualized with ninhydrin. The product is then purifiedby flash column chromatography (18:2:1 isopropanol:methanol:ammoniumhydroxide) to solvent B (18:2:1 isopropanol:methanol:ammoniumhydroxide). The resultant purified products were analysed by ¹H NMR ind₄-methanol.

FIG. 36 shows applications of the strategies described herein for thesynthesis of various unnatural amino acids and their derivatives.

Assay for Biofilm Growth

Stock test solutions were prepared as follows: a 1 mg sample of testcompound was dissolved in 400 uL of DMSO, and diluted to 4 mL in MilliQwater. When diluted 1:50 in the test wells the concentration of atypical unnatural amino acid will be ˜5 ug/mL in 0.2% DMSO. Furthersolutions include Gentamycin (10 mg/mL in MilliQ water) and Tobramycin(0.5 mg/mL in 10% DMSO/MilliQ water).

Each of the following three solutions were autoclaved at 121° C. for 20minutes: “incomplete” M63 Media stock (3 g KH₂PO₄, 7 g K₂HPO₄, 2 g(NH₄)₂SO₄ in 1 L diH₂O; 20% Arginine Solution (6 g Argininein 24 mLdiH₂O); and 1M MgSO₄ solution (2.46 g MgSO₄ diluted to 10 mL withdiH₂O). After autoclaiving, 20 mL 20% autoclaved arginine solution and 1mL autoclaved 1 M MgSO4 solution were added to 1 L of autoclaved“incomplete” M63 stock solution.

Bacterial cultures of P. aeruginosa strain PA14, for example, areprepared from frozen stocks [“Common Virulence Factors for BacterialPathogenicity in Plants and Animals”. Laurence G. Rahme, Emily J.Stevens, Sean F. Wolfort, Jing Shao, Ronald G. Tompkins and Frederick M.Ausubel, Science, Vol. 268, No. 5219 pp. 1899-1902 (1995), Biology(references—O'Toole, et al., Research in Microbiology 162, (2011)680-688; J Vis Exp. 2011; (47): 2437.http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182663/)] by scraping asmall amount from the stock, using the tip of a 100-200 uL steriledisposable pipette, and transferring it to a sterile test tubecontaining 5 mL LB broth (prepared according to directions fromcommercial LB powder, Fisher Scientific, BP1426-500, Lot #104185). Thistube is then incubated on a shaker for approximately 24 hours at 37° C.The resulting bacterial suspension is then diluted 1:100 in complete M63to give the suspension of P. aeruginosa for distribution to theindividual wells in the plate.

For each well plate, in each of the six columns of Row A, six newsynthetic unnatural amino acids were tested. Each of these tests will bereplicated in Row B. Row C (replicated in row D) contained controls:Column 1 and 2, rows C and D, were purified samples of two compounds—oneinactive and one active (negative and positive controls); column 3tested a known antibacterial, (gentamycin); column 4 a known antibioticto treat Pa infections, (tobramycin); column 5, just Pa14 in M63, in thepresence of a final concentration of 0.2% DMSO (the final wellconcentration of DMSO used to solubilize test compounds). (Column 5confirmed that in the absence of any drugs the Pa forms biofilms).Finally, in column 6, rows C and D, media alone (in the presence ofDMSO) to confirm that no biofilm is formed in the absence of bacteria.For those wells containing compounds, 10 uL samples were used.

To stain the biofilm, the wells were rinsed with water, after which 750uL of 0.1% crystal violet stain (in deionized water) was added to eachwell and incubated for 10 minutes. The wells were once again rinsed withwater. Absorbance was then measured using Plate Reader SoftMaxPro at 600nm absorbance. A photograph of a representative example is shown in FIG.31.

Primary Antimicrobial Screening and Hit Confirmation

Samples were prepared in DMSO and water to a final testing concentrationof 32 μg/mL or 20 μM and serially diluted 1:2 fold for 8 times. Eachsample concentration was prepared in 384-well plates, non-bindingsurface plate (NBS; Corning 3640) for each bacterial/fungal strain orTissue-culture treated (TC-treated; Corning 3712/3764) black formammalian cell types, all in duplicate (n=2), and keeping the final DMSOconcentration to a maximum of 0.5% DMSO. All the sample preparation wasdone using liquid handling robots.

For the antibacterial screening, all bacteria were cultured inCation-adjusted Mueller Hinton broth (CAMHB) at 37° C. overnight. Asample of each culture was then diluted 40-fold in fresh broth andincubated at 37° C. for 1.5-3 h. The resultant mid-log phase cultureswere diluted (CFU/mL measured by OD600), then added to each well of thecompound containing plates, giving a cell density of 5×10⁵ CFU/mL and atotal volume of 50 μL. All the plates were covered and incubated at 37°C. for 18 h without shaking.

Fungi strains were cultured for 3 days on Yeast Extract-Peptone Dextrose(YPD) agar at 30° C. A yeast suspension of 1×106 to 5×106 CFU/mL (asdetermined by OD530) was prepared from five colonies. The suspension wassubsequently diluted and added to each well of the compound-containingplates giving a final cell density of fungi suspension of 2.5×103 CFU/mLand a total volume of 50 μL. All plates were covered and incubated at35° C. for 36 h without shaking.

HEK293 cells were counted manually in a Neubauer haemocytometer and thenplated in the 384-well plates containing the compounds to give a densityof 6000 cells/well in a final volume of 50 μL. DMEM supplemented with10% FBS was used as growth media and the cells were incubated togetherwith the compounds for 20 h at 37° C. in 5% CO₂.

Cytotoxicity (or cell viability) was measured by fluorescence, ex:560/10 nm, em: 590/10 nm (F560/590), after addition of 5 μL of 25 μg/mLResazurin (2.3 μg/mL final concentration) and after incubation forfurther 3 h at 37° C. in 5% CO₂. The fluorescence intensity was measuredusing a Tecan M1000 Pro monochromator plate reader, using automatic gaincalculation.

Colistin and Vancomycin were used as positive bacterial inhibitorstandards for Gram-negative and Gram-positive bacteria, respectively.Fluconazole was used as a positive fungal inhibitor standard for C.albicans and C. neoformans. Tamoxifen was used as a positive cytoxicitystandard. Each standard was provided in 4 concentrations, with 2 aboveand 2 below its MIC or CC₅₀ value, and plated into the first 8 wells ofcolumn 23 of the 384-well NBS plates.

The quality control (QC) of the assays was determined by Z′-Factor,calculated from the Negative (media only) and Positive Controls(bacterial, fungal or cell culture without inhibitor), and theStandards. Plates with a Z′-Factor of ≥0.4 and Standards active at thehighest and inactive at the lowest concentration, were accepted forfurther data analysis. For any further information, please refer to theCO-ADD website.

While the novel technology has been illustrated and described in detailin the figures and foregoing description, the same is to be consideredas illustrative and not restrictive in character, it being understoodthat only the preferred embodiments have been shown and described andthat all changes and modifications that come within the spirit of thenovel technology are desired to be protected. As well, while the noveltechnology was illustrated using specific examples, theoreticalarguments, accounts, and illustrations, these illustrations and theaccompanying discussion should by no means be interpreted as limitingthe technology. All patents, patent applications, and references totexts, scientific treatises, publications, and the like referenced inthis application are incorporated herein by reference in their entirety.

We claim:
 1. A compound, wherein the compound is at least one enantiomerof at least one compound selected from the group consisting of:

or a pharmaceutically acceptable salt thereof, or a metabolite thereof,or wherein the compound is,

or a pharmaceutically acceptable salt thereof, or a metabolite thereof.2. A method for reducing the growth of a microorganism, comprising thesteps of: treating one or more microorganisms with at least one compoundselected from the compounds according to claim 1, wherein the one ormore microorganisms is gram-negative bacteria.
 3. The method accordingto claim 2, wherein the one or more microorganisms is Pseudomonasaeruginosa, Candida albicans, and/or Cryptococcus neoformans.
 4. Themethod according to claim 2, further comprising the step of: treating anarea that has been infected by the one or more microorganisms.
 5. Themethod according to claim 4, wherein the area comprises surfaces or hairof an animal, a human, or a plant.
 6. A method of treating a patient,comprising the steps of: providing to a patient having a gram-negativemicrobial infection at least one therapeutically effective dose of atleast one compound selected from the compounds according to claim
 1. 7.The method according to claim 6, further comprising the step of:diagnosing a patient with microbial infections, wherein the microbialinfections are caused by Pseudomonas aeruginosa, Candida albicans,and/or Cryptococcus neoformans.
 8. The method according to claim 6,wherein the therapeutically effective dose is on the order of betweenabout 1 mg/kg to about 7 mg/kg and the dose of the compound isadministered to the patient at least once per day.
 9. The methodaccording to claim 6, wherein the therapeutically effective dose is onthe order of between about 3 mg/kg to about 5 mg/kg and the dose of thecompound is administered to the patient at least once per day.
 10. Themethod according to claim 6, wherein the therapeutically effective doseis administered by intravenous or intramuscular injections.
 11. Thecompound according to claim 1, wherein the compound is at least oneenantiomer of at least one compound selected from the group consistingof:

or a pharmaceutically acceptable salt thereof, or a metabolite thereof.12. The compound according to claim 1, wherein the compound is at leastone enantiomer of at least one compound selected from the groupconsisting of:

or a pharmaceutically acceptable salt thereof, or a metabolite thereof.13. The compound according to claim 1, wherein the compound is

or a pharmaceutically acceptable salt thereof, or a metabolite thereof.